Growth-promoting proteins and peptides for kidney epithelial cells

ABSTRACT

Novel growth peptides derived from protein factors having molecular weights of about 22 and 45 kDa stimulate mitogenic activity of epithelial, but not fibroblastic cells, in particular, kidney epithelial cells. A source of the factors is scrape-wounded kidney epithelial cells in culture. Synthetic peptides having sixteen amino acids or less, in particular a hexapeptide, YPQGNH (SEQ ID NO: 2) maintain the mitogenic activity. The peptide AQPYPQGNHEASYG (14-Ser) (SEQ ID NO: 15) is effective in reversing acute renal failure in animals. The growth-promoting characteristics of the 22 and 45 kDa proteins and the peptides are useful in treating and diagnosing patients with kidney disease. Nucleotide sequences that encode the factor are useful to develop probes to locate similar factors, to identify genetic disorders involving the factor, and to produce the factor by genetic recombinant methods. The nucleotide sequences and fragments thereof, are also useful for diagnosis and treatment of kidney disorders.

The United States government may have rights in the disclosed inventionbecause of partial support under National Institutes of Health #DK39689.

BACKGROUND

Novel growth peptides derived from protein factors having molecularweights of about 22 and 45 kDa stimulate mitogenic activity ofepithelial, but not fibroblastic cells, in particular, kidney epithelialcells.

Acute renal failure is a serious disease associated with high mortalityfor which no "real" treatment currently exists. Acute renal failure isdefined as the abrupt disruption of previously normal kidney function.It is caused by a wide variety of mechanisms including circulatoryfailure (shock), vascular blockade, glomerulonephritis, and obstructionto urine flow. In addition it can occur following surgery, trauma,sepsis, or with certain medications, particularly antibiotics andanticancer agents.

In 1985 some 140,000 Americans were hospitalized with acute renalfailure (see 1990 Long Range Plan). The average cost of treatmentassociated with these cases was over $9000. Based on the growth in thedisease over the past several years and normal inflation, it wasestimated that currently some 240,000 patients develop acute renalfailure annually at a cost of over $10,000 per patient. That translatedto a staggering total cost to the U.S. healthcare system of almost $2.5billion per year.

                  TABLE 1                                                         ______________________________________                                        AVERAGE COST PER HOSPITAL DISCHARGE FOR KIDNEY                                AND UROLOGIC DISEASES, UNITED STATES, 1985.sup.1                                               Number of                                                                             Average Cost per                                                      Discharges                                                                            Discharge                                            ______________________________________                                         1. Acute renal failure                                                                          139,134   $9,329                                            2. Chronic renal failure                                                                        395,066   9,249                                             3. Kidney disease of diabetes mellitus                                                          96,731    6,819                                             4. Kidney cancer  47,384    6,145                                             5. Hypertensive renal disease                                                                   182,625   5,796                                             6. Other intrinsic/systemic diseases                                                            79,683    5,061                                             7. Bladder cancer 125,108   4,758                                             8. Impotence      30,452    4,344                                             9. Prostate cancer                                                                              246,201   3,791                                            10. Testicular cancer                                                                            14,219    3,711                                            11. Benign prostatic hyperplasia                                                                 482,348   3,648                                            12. Polycystic kidney disease                                                                    44,155    3,213                                            13. Glomerulonephritis                                                                           79,531    3,135                                            14. Bladder disorders                                                                            342,211   3,064                                            15. Urinary stone disease                                                                        453,018   2,920                                            16. Urinary tract infection                                                                      1,583,309 2,549                                            17. Incontinence   162,574   2,547                                            18. Hematuria      173,495   2,375                                            19. Prostatitis    108,024   2,010                                            20. Obstructive uropathy.sup.2                                                                   397,074   1,842                                            21. Other genitourinary infections                                                               147,215   1,339                                            22. Preeclampsia   139,000   1,025                                            23. Testicular dysfunction                                                                       7,019     950                                              ______________________________________                                         .sup.1 Includes payments to physicians.                                       .sup.2 Includes vesicoureteral reflux.                                        SOURCES:                                                                      National Center for Health Statistics: National Hospital Discharge Survey     i985 (all listed diagnoses). Department of Veterans Affairs, for year         ending September 30, 1986 (firstlisted diagnoses) (unpublished). Health       Care Financing Administration, Medicare provider analyses and review data     1985 (unpublished).                                                      

As can be seen in Table 1 from the Plan, kidney disease contributes tomajor medical costs in the United States, so factors reducing time torecovery, are beneficial to society.

Equally significant, is the fact that the number of cases of acute renalfailure is growing at a rate of 9% per year (NIH, 1995) and this highrate of growth is expected to continue. A reason given for this rise inthe incidence of renal failure is that "sicker" patients with a highrisk of renal failure are surviving longer.

1. Older patients, who have a significantly higher incidence of acuterenal failure (e.g., patients over 65 are 5 times more likely to behospitalized for acute renal failure than those ages 45 to 64) are nowsurviving serious medical incidents (e.g., heart attack, stroke) as wellas complicated surgery. Improved hospital intensive care units with moresophisticated monitoring and life support systems also aid in keeping"sicker" patients alive. In addition improved therapeutic agents fortreating cancer and life-threatening infections are often nephrotoxic.

2. Neonates, who have an extremely high risk of kidney failure are alsosurviving at shorter terms and at significantly lower birth weights.Such infants formerly had difficulties overcoming severe lung and heartproblems, but these problems can now be successfully treated withimproved drugs and techniques, particularly in specialized neonatalintensive care units.

Because these advances in treatment modalities are expected to continueand even accelerate, it is likely that the number of cases of acuterenal failure will continue to increase, perhaps at an even faster rate.

At the present time no real "cure" exists for acute renal failure. Thecurrent method of treatment is to "rest" the kidney by performingdialysis to correct metabolic imbalances and wait for kidney function toreturn spontaneously.

Dialysis is a technique in which impurities and toxins from the blood,that are normally cleared through the kidneys are artificially removedthrough an extra-corporeal circuit and filter (hemodialysis) or throughthe peritoneal membrane. By removing such impurities the lifethreatening metabolic imbalances resulting from kidney failure can becorrected and the patient stabilized.

Mortality rates resulting from a patient's developing acute renalfailure are extremely high. A recent study (Levy et al., 1996) thatanalyzed the effect of acute renal failure on patient mortality citessuch rates as ranging from 42% to 88% based on 18 previously publishedreports. These rates have remained essentially unchanged since the early1950's. In the 1996 study itself the mortality rate for hospitalizedpatients who developed acute renal failure was 5 times higher comparedto similar patients without renal failure (34% vs. 7%).

A key finding of this study is that "acute renal failure appears toincrease the risk of developing severe non-renal complications that leadto death and should not be regarded as a treatable complication ofserious illness." Thus it appears that the rapid reversal of acute renalfailure can significantly reduce the risk of mortality in patients whoalso frequently have complicated clinical courses by preventing thedevelopment of severe and often fatal non-renal complications.

It has long been known that the kidney is one of the few human organsthat has an ability to repair itself after injury. Even in cases wherethe kidney has been irreversibly damaged, and there is extensivenecrosis of kidney cells, strong evidence exists that some new cellgrowth occurs.

It has been proposed that growth factors are a therapeutic approach tostimulate or augment the regenerative process in the injured kidney andthereby reduce the severity and shorten the course of acute renalfailure. The use of growth factors as a treatment for acute renalfailure was first proposed by Toback (1984). However, finding suitablegrowth factors proved difficult. The rationale for this strategy wassubsequently expanded after several specific growth factor proteins wereidentified (Mendley and Toback, 1989; Toback 1992 a and b). However, nofactors have yet been confirmed as useful in treating humans.

Growth factors acting in vivo to stimulate proliferation and migrationof noninjured tubular cells in the kidney, and possibly to facilitaterecovery of sublethally-injured cells as well, would be beneficial. Aspecific growth factor could be used in combination with sufficientnutrients, calories, and dialytic therapy to increase survival ofpatients with renal problems. For example, administration of growthfactors could (1) increase positive outcomes in patients with cadavericrenal transplants, a situation in which acute renal failure isassociated with increased rejection, (2) shorten the duration of acuterenal failure which would increase patient survival, and (3) reduce thenumber of days required for hemodialysis treatment during the renalfailure syndrome.

Autocrine growth factors are produced locally by the same cells on whichthey act. They appear to be produced in response to a stimulating eventsuch as cell injury. Moreover, they are produced in extremely smallquantities and may exist at detectable levels for only a short time.Consequently, they have been quite difficult to isolate and identify.

Two other types of growth factors--paracrine and endocrine--both appearto have some role in stimulating kidney cell growth. Paracrine factorsact on adjacent cells (rather than on themselves) while endocrinefactors are produced in one cell and transported (e.g., by the bloodstream) to act on another, distant cell. Several of these types offactors, which are typically produced in larger quantities, and have alonger "half life" than some autocrine factors, have been discovered,and their cDNAs identified.

Animal and Clinical Studies

Several growth factors have been studied in an acute renal failure ratmodel to determine their efficacy in speeding recovery. The results ofthese studies give encouraging support to the theory that growth factorsmay play a major role in accelerating kidney repair. Three of the mostimportant of these are:

1. Epidermal growth factor (EGF) The EGF factor has been reported toaccelerate recovery in rats with acute renal failure. However, it wasnoted that EGF also mobilizes calcium from bone, which is a serious sideeffect that will likely prohibit its use in humans.

2. Insulin like growth factor -1 (IGF-1). Several studies in the ratmodel confirm that this factor is indeed efficacious. However, in twoclinical studies in humans IGF-1 did not appear to have any substantialeffect in speeding a patient's recovery from acute renal failure.

3. Osteogenic protein -1 (OP-1) is a bone growth factor already approvedfor human use in repairing bone, cartilage, and eye tissue. AlthoughOP-1 may play a key role in the embryonic development of human kidneys,it is not clear how it works to help repair adult kidney cells. It ispossible that OP-1 and other autocrine kidney growth factors togethercould have complementary mechanisms of action.

Autocrine Kidney Growth Factors

Although the animal study results on the previously identified growthfactors are encouraging, none of these factors are used clinically atpresent. Of particular note is that the kidney messenger RNA for thethree growth factors described above--EGF, IGF-1 and OP-1-actuallydecreases in the kidneys during acute renal failure. Logically, if agrowth factor is to be effective in repairing injury and reversing acuterenal failure, its levels would be expected to increase during thisclinical event.

Some of the factors already identified are released by kidney epithelialcells and are capable of stimulating growth of the cells in an autocrinemanner. For example, monkey kidney (BSC-1) cells respond to culturemedium with a reduced concentration of potassium by releasing the "LowPotassium Growth Factor," and respond to a reduced concentration ofsodium by releasing the "Low Sodium Growth Factor" (Mordan and Toback,1984; Walsh-Reitz et al., 1986; Toback et al. 1992b and 1995).

A significant need exists for new therapeutic approaches to "cure," orat a minimum, speed the reversal of acute renal failure.

SUMMARY OF THE INVENTION

This invention is directed to growth promoting proteins and peptides,initially referred to as a protein Wound Growth Factor (WGF) because ofthe manner of production in culture of the basic factor. Furtheranalysis revealed factors of two molecular weights,

    __________________________________________________________________________    1                     12   16                                                 22 kDa isoform: NH.sub.2 - A Q P Y P Q G N H E A                                                    T  S S S F --COOH (SEQ                                  ID NO: 4)                                                                     1                     12  14                                                  45 kDa isoform: NH.sub.2 - A Q P Y P Q G N H E X                                                    A/S                                                                              Y G --COOH (SEQ ID NO:                               1)                                                                            __________________________________________________________________________

Peptides of various lengths are within the scope of the presentinvention, as long as the mitogenic hexamer sequence, NH₂-tyrosine-proline-glutamine-glycine-(YPQGNH)(SEQ ID NO: 2) is included.

An embodiment of a novel peptide is a potent mitogen for monkey kidneyepithelial cells in culture 14 amino acids, AQPYPQGNHEASYG (14-Ser)(SEQID NO: 15). Compared to other known renal growth factor mitogens, thispeptide has a mitogenic effect that is either additive with, equivalentto, or more potent than other known factors: e.g., epidermal growthfactor, acidic fibroblast growth factor, basic fibroblast growth factor,insulin-like growth factor, vasopressin, or calf serum.

The native factor (WGF) is released into culture medium when a kidneycell monolayer is subjected to mechanical scrape wounding with a pipettetip. This was a novel finding. That is, the growth factor is releasedfrom scrape wounded kidney cells and can stimulate proliferation of thecells. Thus, it is an autocrine growth factor. A source of cells whichreleases the factor is the BSC-1 cell line (nontransformed African greenmonkey kidney epithelial cells). (ATCC CCL 26 8S-C-1) "Bioactive WGF" isdefined herein as a factor that stimulates mitogenic activity incultured renal cells and is generally what is meant by "WGF" herein."WGF" includes several peptides with mitogenic activity. These peptidesmay be synthesized by techniques well known to those of skill in theart, including recombinant genetic technology. A preferred peptide has14 amino acids, the sequence is AQPYPQGNHEASYG (14-Ser)(SEQ ID NO: 15).

The appearance of growth-promoting activity after wounding seems to bemediated by the proteolytic activation of an inactive precursor of WGF.Evidence for this is that preincubation of the cells for 10 minutes witheach of the following diverse protease inhibitors prevented theappearance of growth-promoting activity after wounding: aprotinin,phenylmethylsulfonyl-fluoride(Sigma) (PMSF), antipain,L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone-hydrochloride (TLCK) or.sub.α2 -macroglobulin. None of these agents inhibited cell growth whenadded to cells of nonwounded cultures. When added to the medium afterthe appearance of WGF mitogenic activity, neither PMSF nor aprotininappeared to inhibit the increment in cell proliferation. HPLC-purifiedWGF does not appear to be a protease because it did not exhibitproteolytic activity when assayed using Protease Substrate Gel tablets(BioRad).

WGF exhibits a growth-promoting activity that is released into theculture medium of BSC-1 cells. Isolation and purification of componentsresponsible for this growth-promoting activity reveals that it behaveson sodium dodecylsulfate (SDS)-polyacrylamide electrophoresis as if ithas a relative molecular mass (Mr) of 22 and/or 45 kilodaltons (kDa).WGF is a protein that is a mitogen for monkey kidney BSC-1 cells, butnot for 3T3 fibroblasts. Release of WGF also appears to be relativelykidney epithelial cell-type specific in origin because it appears afterwounding BSC-1 cells in culture, but not after wounding fibroblasts inculture.

Therefore, an aspect of the present invention is a protein designated"WGF" having the following characteristics:

a) an estimated molecular weight of about 45 and/or 22 kDa, saidestimate obtained by electrophoresing the HPLC-purified protein on anSDS-polyacrylamide gel;

b) capability of stimulating mitogenic activity when in contact withcultured cells; and

c) released by BSC-1 cells in culture by scrape wounding.

In particular, the protein has a partial amino acid sequence at itsamino terminal end as follows: NH₂-alanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-X-alanine/serine-tyrosine-glycine-COOH (SEQ ID NO: 1). (X=undefinedamino acid)

Another WGF protein has an estimated molecular weight of about 22 kDa,said estimate obtained by electrophoresing the HPLC-purified protein onan SDS-polyacrylamide gel and a partial amino acid sequence at its aminoterminal end as follows: NH₂-alanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-alanine-threonine-serine-serine-serine-phenylalinine-COOH (SEQ IDNO: 4).

A protocol suitable to purify WGF from conditioned cell culture mediumutilizes ultrafiltration, heparin-affinity chromatography andreversed-phase (RP) high-performance liquid chromatography (HPLC). 6,400fold purification is achieved, although the yield of WGF protein isextremely low, usually in the range of 50 ng protein per liter ofconditioned medium.

The size of bioactive WGF was defined by electrophoresing HPLC-purifiedWGF on SDS gels in parallel with standard proteins (i.e., proteins ofknown sizes), slicing the gel into 2-mm wide gel fragments, eluting eachfragment in buffer, and then assaying the eluate for mitogenic activityusing cultures of BSC-1 cells. This experimental strategy indicated thatWGF proteins have an estimated M_(r) of 22 and 45 kDa and are mitogenic.

Generally "proteins" is the term used for molecules of about 50 aminoacids or greater. Peptides are smaller.

A single sharp peak of absorbing material at 214 nm obtained by RP-HPLCexhibits growth-promoting activity on kidney epithelial but notfibroblastic cells, and yields several bands on SDS-polyacrylamide gelelectrophoresis following silver staining.

Amino acid compositional analysis of material that formed the sharp peakconfirmed the protein character of WGF. Microsequencing revealed thefirst 16 amino acids of the amino (NH₂) terminus of the 22 kDa isoform:NH₂-alanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-alanine-threonine-serine-serine-serine-phenylalanine-COOH (SEQ IDNO: 4). For the 45 kDa isoform 14 amino acids at the amino-terminus havebeen identified: NH₂-alanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-X-alanine/serine-tyrosine-glycine-COOH (SEQ ID NO: 1). The identityof the amino acid at position 11 is uncertain (X), and it is notpossible to determine whether an alanine (A) or serine (S) is atposition 12. A search of the seven peptide sequence databases in theExperimental GENINFO(R) BLAST Network Service (Blaster) operated by theNational Center for Biotechnology Information (NCBI) indicated that theamino-terminal sequences are that of novel proteins.

Of substantial importance is the additional finding that peptidessmaller than the full preferred 16 amino acid sequence also have strongmitogenic specific activity. This finding is quite significant becausethese small peptides (1) are much less likely to be antigenic (i.e.,they can be directly infused into another animal or human without beingrejected by the immune system) and (2) can be readily prepared in largequantities and modified using a peptide synthesizer without first havingto find a cDNA clone that encodes the entire 22 or 45 kilodaltonprotein, and then expresses the recombinant protein.

In addition to producing the factor by wounding cultured cells,synthetic peptides are produced that contain the mitogenic activity. Ofparticular interest is that a synthetic peptide whose sequence is basedon the first eleven amino acid residues of the 22 kDa protein exhibitsmitogenic activity. Moreover, other polypeptides that are short peptidedomains of the factor are also within the scope of the presentinvention. An hexamer was the smallest peptide which still maintainedmitogenic activity (YPQGNH) (SEQ ID NO: 2).

A peptide comprising an amino acid sequence of NH₂-tyrosine-proline-glutamine-glycine-asparagine-histidine-COOH (SEQ IDNO: 2) is suitable for the practice of the invention. Generally, thepeptide has a length of from 7 to 16 amino acids but other lengths arealso suitable if the mitogenic and/or antigenic function is preserved.

Both transforming growth factor-beta 2, and a synthetic 5-amino acidpeptide having the sequence YPQGN (SEQ ID NO: 3) block the mitogeniceffect of AQPYPQGNHEASYG (SEQ ID NO: 15). The glycosaminoglycans,heparin and keratan sulfate, each potentiate the mitogenic effect of thepeptide.

A peptide having the sequence AQPYPQGNHEASYG (SEQ ID NO: 15) and otherrelated peptides derived from the NH₂ -termini of Wound Growth Factorisoforms, were evaluated for their capacity to alter the course of acuterenal failure (ARF) in a nephrotoxic rat model; this syndrome commonlyafflicts humans.

Mercuric chloride given subcutaneously (s.c.) was used to induce ARF inrats, and a solution of each peptide was evaluated for its capacity toenhance survival and speed recovery of renal function, assessed bymeasuring the serum creatinine concentration during the ensuing 7 days.

Administration of a peptide having the sequence AQPYPQGNHEASYG (SEQ IDNO: 15), s.c. 1 hour after administration of mercuric chloridesignificantly improved recovery of renal function two days later andimproved survival after three days. Similar beneficial effects of thepeptide on survival and recovery of renal function were observed when itwas administered 24 hours before induction of the ARF syndrome.

Evidence that the peptide improved survival and recovery of renalfunction by stimulating DNA synthesis in cells near the site of mercuricchloride-induced renal injury was obtained by using bromodeoxyuridine tolabel DNA in the regenerating kidneys. The peptide was more potent thanepidermal growth factor, an agent of known efficacy in this modelsystem, in promoting survival when given 24 hours before renal injury,and equivalent when given shortly thereafter. Improved survival and amore rapid recovery of renal function in response to treatment with thepeptide are expected in humans with nephrotoxic as well as ischemic ARF.

Suitable peptides for the practice of the invention include:

AQPYPQGNHEATSSSF (SEQ ID NO: 4);

AQPYPQGNHEATSSS (SEQ ID NO: 5);

AQPYPQGNHEA (SEQ ID NO: 6);

AQPYPQGNHEAT (SEQ ID NO: 7);

AQPYPQGNHEATS (SEQ ID NO: 8);

AQPYPQGNHEATSS (SEQ ID NO: 9);

AQPYPQGNHEATSY (SEQ ID NO: 10);

AQPYPQGNHEAAYG (SEQ ID NO: 11);

AQPYPQGNHEAAY (SEQ ID NO: 12);

AQPYPQGNHEAA (SEQ ID NO: 13);

AQPYPQGNHE (SEQ ID NO: 14);

AQPYPQGNHEASYG (SEQ ID NO: 15);

AQPYPQGNHEASY (SEQ ID NO: 16);

AQPYPQGNHEAS (SEQ ID NO: 17);

QPYPQGNHEA (SEQ ID NO: 18);

AQPYPQGNH (SEQ ID NO: 19);

QPYPQGNHE (SEQ ID NO: 20);

PYPQGNHEA (SEQ ID NO: 21);

QPYPQGNH (SEQ ID NO: 22);

PYPQGNHE (SEQ ID NO: 23);

YPQGNHEA (SEQ ID NO: 24);

PYPQGNH (SEQ ID NO: 25); and

YPQGNHE (SEQ ID NO: 26);

YPQGNHEATSSSF (SEQ ID NO: 27);

YPQGNHEATSSS (SEQ ID NO: 28);

YPQGNHEATSS (SEQ ID NO: 29);

YPQGNHEATS (SEQ ID NO: 30); and

YPQGNHEAT (SEQ ID NO: 31).

A mitogenic fragment of protein is the peptide YPQGNH (SEQ ID NO: 2); apeptide having the sequence AQPYPQGNHEASYG (SEQ ID NO: 15) is preferred.

A composition comprising the proteins or peptides described herein iswithin the scope of the invention.

A method for producing a protein or peptide of the present inventionincludes the steps of:

a) culturing kidney epithelial cells in media,

b) wounding the cells in culture, and

c) obtaining the protein from the conditioned media.

The protein obtained is isolated from the conditioned media andpurified. A source of the kidney epithelial cells in culture is theBSC-1 African green monkey kidney epithelial cell line.

A recombinant DNA method of making a protein or peptide of the presentinvention includes the following steps:

a) obtaining a nucleotide sequence encoding the protein or peptide; and

b) using the nucleotide sequence in a genetic expression system to makethe protein or peptide.

The peptide can also be prepared directly on a peptide synthesizerwithout recourse to cellular or molecular biological techniques.

An antibody to WGF is an aspect of the present invention. Availabilityof the antibody provides a diagnostic tool to measure the amount of thefactor in urine, blood and tissue. New diagnostic insights arefacilitated in patients receiving drugs with nephrotoxic potentialduring treatment of infections or malignancies, and in individuals withrenal injury or neoplasia. Such an antibody may also be used to detectrenal cancer in the remnant kidneys of patients undergoing chronicperitoneal and hemodialysis. The antibody is directed to a protein orpeptide including the active site, that is, generally includes YPQGNH(SEQ ID NO: 2).

A diagnostic kit is used to measure the quantity of a WGF protein or amitogenic peptide therefrom, in a biological sample to detect acuterenal injury or the early onset of kidney disease, monitor treatment ofrenal cell cancer, or recognize the conversion of benign renal cysts inchronic dialysis patients to carcinomas or cystadenocarcinomas. The kitincludes in separate containers:

a) an antibody to WGF or to a mitogenic peptide therefrom; and

b) a means for detecting a specific complex between the WGF protein or amitogenic peptide and the antibody.

The invention includes the use of the protein in preparing a compositionfor medical treatment of kidney disease, said preparation comprisingobtaining the protein and adding to it a suitable carrier.

The new kidney growth factor proteins and peptides of the presentinvention and antibodies directed to them have diverse uses in clinicalmedicine. The WGF peptides are useful for stimulating kidney cellgrowth, a characteristic useful for treatment of acute renal failure. Itis particularly desirable to speed recovery in patients with acute renalfailure, especially those receiving cadaveric kidney transplants.Infusion of the protein into patients is directed to shortening theduration of the acute renal failure episode which would increase patientsurvival, and reduce the number of days required for hemodialysistreatment during the renal failure syndrome. The peptides also providean in vitro standard of comparison for other candidate growth factors.

It is expected that WGF will have a role as a therapeutic agent to slowthe progression of established kidney diseases such as chronicglomerulonephritis or interstitial nephritis. WGF and its receptorappears to be on the surface of renal epithelial cells. If WGF is foundto be a ligand for receptors on the surface of specific renal epithelialcell types along the nephron, it is to be considered in cancerchemotherapy. If it is found to be cancer cell-type specific, the growthfactor could be conjugated with a cellular toxin, a radioactive isotope,or cytotoxic antibody to produce powerful new chemotherapeutic agents.

A method of treating a person with acute renal failure, includes: a)preparing a pharmacologically effective amount of native WGF protein orWGF-derived peptide in a suitable diluent; and b) administering thepreparation to the person. The WGF may be ligated to a cytolytic ligand,e.g. a toxin.

The invention also relates the use of a protein or peptide describedherein to obtain a composition useful in treating a person with acuterenal failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates growth-promoting activity of wound growthfactor-derived peptides on growth of BSC-1 cells.

FIG. 2 illustrates enhanced survival FIG. 2A and renal functionalrecovery (FIG. 2B) of rats given a WGF-derived peptide (14 Ser) aftermercuric chloride-induced acute tubular necrosis. (FIG. 2B: Values aremeans±standard error. *, Student's t-test, P<0.034.) (FIG. 2A: Percentsurvival is number of rats alive divided by total number alive and dead;*, chi squared, P<0.050.)

DETAILED DESCRIPTION OF THE INVENTION Production Of The Wound GrowthFactor

Confluent monolayer cultures were mechanically scraped using a 200 μLpipet tip (Continental Laboratory Products, San Diego, Calif.). Whenobserved under a microscope, this scraping did not appear to damage thecells, only to cause them to separate from each other and retract,leaving a narrow path in the culture dish that could be seen with thenaked eye. (This process of scraping the cells has been termed"wounding" and has been used for many years as a model system to studyrepair of corneal abrasions of the eye. In effect, pressure is appliedto cells sufficient to disrupt intercellular adhesion.) To determinewhether kidney epithelial cells released an autocrine factor into themedium after wounding, conditioned culture medium was removed from theculture dish after scrape-wounding a monolayer of BSC-1 cells. Analiquot was assayed for any growth-promoting activity on a nonwounded"detector" culture (1.2×10⁶ cells per 55-mm dish).

Growth Promoting Activity of the Factor

Growth-promoting activity in an aliquot of the conditioned medium wasassayed by counting the number of cells in a detector culture four dayslater and comparing the number to that in a control culture to which analiquot of medium from a nonwounded culture had been added. Thisstrategy showed that wounded kidney epithelial cells of BSC-1 linereleased a growth-promoting activity that was initially termed "WoundGrowth Factor." 3T3 fibroblasts did not produce a similar effect.

For the assay, cells were detached from the dish with a solution oftrypsin and an aliquot was counted in a hemocytometer. Confluentcultures were used for this assay so that cells exposed to WGF had acell count of 2.6×10⁶ cells/culture, whereas cells treated with analiquot of medium from a non-wounded culture had a count of 2.0×10⁶cells four days after the additions. Thus WGF stimulated cellproliferation by 30% in this assay. When high-density, quiescentcultures (3×10⁶ cells/55-mm dish) were used to assay mitogenic activityby measuring [³ H] thymidine incorporation into DNA, WGF enhanced DNAsynthesis about by 60%. To prepare WGF, cells can be scrape-woundedevery other day, yet maximal activity will be released into theconditioned medium. WGF is mitogenic for both sparse and confluentcultures of BSC-1 cells, and is stable upon storage at 4° C. to -40° C.for many weeks.

Characterization of Wound Growth Factor Mitogenic Activity

After detection of growth-promoting activity in the conditioned mediumof scrape-wounded cultures of BSC-1 cells, efforts to determine size andcomposition of the mitogenic factor were undertaken. Initially, filtershaving different molecular weight cut-offs were utilized to show thatthe growth-promoting activity passed through an Amicon YM membranehaving a cut-off of 100 kDa, but was retained by a membrane with acut-off of 30 kDa, suggesting the size of the mitogenic factor wasgreater that 30 kDa, but less than 100 kDa. These membranes provide onlya very crude estimate of size of the mitogenic factor.

Experiments strongly suggested that the activity was a protein becauseit could be destroyed by exposure to trypsin (100 μg/ml for 3 hours),dithiothreitol (65 mM for 1 hour), acetic acid (1 M for 5 hours), or byheat (70° C. for 20 minutes).

Characterization of the net electrical charge on WGF was sought usingDEAE and CM cellulose matrices (Pharmacia) that have different charges.Results indicated that WGF was cationic. Subsequent experimentsindicated that it bound tightly to a heparin cartridge (Pharmacia HiTrap Heparin) from which it could be eluted with 0.4 to 1.0 M sodiumchloride.

Pentosan polysulfate (Sigma Chemical, St. Louis) which is known to blockthe activity of heparin-binding growth factors such as acidic and basicfibroblast growth factors also blocked activity of WGF suggesting thatit is a cationic molecule. Studies on a C₄ -reversed-phase HPLC column(Vydac) revealed that the protein was markedly hydrophobic in that aconcentration of 55% acetonitrile (J. T. Baker, HPLC grade) was requiredto elute WGF-growth-promoting activity from the column. WGF activity wasalso stable in 0.1% trifluoroacetic acid (TFA) (J. T. Baker, HPLC grade)and isopropanol.

Certain mitogenically-active fractions of WGF eluted from a C₄reversed-phase HPLC column also bind to concanavalin A-Sepharose 4B(Pharmacia LKB), and are eluted by alpha-methyl mannoside, suggestingthe factor contains carbohydrate. Four fractions from the HPLC columnthat exhibited maximal growth-promoting activity, including the one thatelutes at 55% acetonitrile designated as WGF, were each exposed toconcanavalin A-Sepharose and subsequently to alpha-methyl mannoside. Theeluate was then assayed for its growth-promoting activity. Fractionsthat eluted from the HPLC column at 55% and at 49% acetonitrile bound toconcanavalin A-Sepharose, were eluted by alpha-methyl mannoside and werefully active. In contrast, fractions that eluted at 46% or 57%acetonitrile, or pure epidermal growth factor used as a control mitogenthat is not a glycoprotein did not bind to concanavalin A. These resultssuggest that native WGF is a glycoprotein

WGF, Heparin, and Other Growth Factors

Although scrape wounding of a different type of cells, endothelialcells, releases acidic fibroblast growth factor (aFGF), characterizationof kidney cell WGF indicated that WGF differed from aFGF. Evidence forthis is as follows: (1) heparin (Sigma) augments the mitogenic activityof WGF but inhibits aFGF when each component is added to BSC-1 cells,(2) keratan sulfate (a glycosaminoglycan component of the extracellularmatrix) stimulates mitogenic activity of WGF but has no effect onactivity of aFGF, (3) the mitogenic effects of maximal concentrations ofWGF and aFGF are additive, and (4) the size of WGF is about 45 kDawhereas aFGF is about 16 kDa.

WGF also differs from basic FGF (bFGF) in that (1) bFGF (Gibco/BRL)elutes from an heparin affinity column at 1.5 to 1.6 M NaCl whereas WGFelutes at 0.4 to 1.0 M NaCl, and (2) the mitogenic activity of bFGF is90% inhibited by 55% acetonitrile/0.1% (TFA) which has no effect on theactivity of WGF. Furthermore, aFGF and bFGF mRNA are not detected byNorthern blotting of BSC-1 cells, indicating that genes encoding thesegrowth factors are not expressed in the cells.

The mitogenic effects of maximal concentrations of partially-purifiedWGF and epidermal growth factor (EGF, Promega) are also additive,indicating that they probably exert their mitogenic effects by differentreceptors and signaling pathways.

Treatment of a confluent monolayer of BSC-1 cells with the enzymeheparinase I (Sigma), but not heparinase III (Sigma), releases WGFmitogenic activity from cells into the culture media. However, thisseems to deplete WGF, at least temporarily. When cells are first treatedwith heparinase I, rinsed, the media aspirated, and the monolayer isthen scrape-wounded, no growth-promoting activity is detected in theconditioned media. These observations suggest a role for a heparin-likemolecule such as a glycosaminoglycan that mediates the association ofWGF to the plasma membrane from which it is released upon wounding ortreatment with heparinase I.

If WGF resides on the cell surface, it appears to be protected fromdegradation by trypsin, possibly by carbohydrate residues linked to itsprotein backbone, and/or by associating with glycosaminoglycans adherentto the plasma membrane (glycocalyx). Evidence in favor of thisformulation is derived from the following experiment. A cell monolayerwas exposed to the proteolytic enzyme trypsin (1 or 10 μg/ml) for 10minutes. The enzyme was previously shown to have the capacity to destroyWGF activity. Then soybean trypsin inhibitor (10 μg/ml) was added toneutralize the enzyme. When the cell monolayer was subsequently wounded,full biologically active WGF was released into the culture mediumindicating that exogenous trypsin had not destroyed growth factorassociated with the cells.

WGF does not appear to be stored by the cells in extracellular matrix(ECM). This conclusion is based on the following experiment. A confluentmonolayer of BSC-1 cells is detached from ECM by addition of EGTA (J. T.Baker) to the medium, leaving a coating of ECM on the surface of theculture dish. Fresh medium was added to the dish and its ECM coating wasthen subjected to scrape-wounding. No mitogenic activity was detected inthe conditioned medium, suggesting that the activity resided on orwithin the cells and not in the ECM.

The mitogenic potency of partially-purified WGF is equivalent to themitogenic potency of about 5% calf serum, or 20 pg/ml aFGF, or 20 pg/mlbasic FGF, or 15 ng/ml EGF, using assays disclosed herein.

Comparison of Known Growth Factors to WGF-Derived Peptide; AdditiveEffects

The mitogenic potency of the 14-amino acid peptide AQPYPQGNHEASYG (SEQID NO: 15) having a serine residue at position 12 (14-Ser) was comparedto growth factors and other mitogenic signals for monkey kidneyepithelial cells of the BSC-1 line. The previously identified maximalmitogenic concentration of each growth-promoting signal was employed,and compared to the WGF-derived peptide at concentrations from 0 to 0.5μg/ml.

The following peptide and ionic mitogenic signals were studied:epidermal growth factor (EGF), 50 ng/ml; insulin-like growth factor-I(IGF-I), 50 ng/ml; acidic fibroblast growth factor (FGF-1), 100 pg/ml;basic fibroblast growth factor (FGF-2), 1,000 pg/ml; vasopressin, 75pg/ml; calf serum (0-1%); high potassium medium (5 mM added KCl, finalconcentration 10.4 mM), high sodium medium (25 mM of NaCl added, finalconcentration 180 mM).

Nontransformed monkey kidney epithelial cells of the BSC-1 line weregrown to confluence in Dulbecco's modified Eagle's medium containing 1%calf serum and 1.6 μM biotin in 55-mm culture dishes at 38° C. in a CO₂incubator. When cells achieved a density of about 10⁶ per dish, thespent medium was removed and replaced with fresh medium containing 0.5%calf serum and the 14-amino acid peptide (0.5 μg/ml). Additions of eachof the mitogenic signals at maximal concentration were made to theculture medium. Four days later the number of cells in each culture wascounted in an hemocytometer. Values obtained were the mean of 3 separatecultures. Doubling the calf serum concentration from 0.5% to 1.0% didnot significantly alter the growth of BSC-1 cells. Addition of the14-Ser peptide (0.5 μg/ml) stimulated growth by 25-30% at each of theseserum concentrations.

1. Comparisons with EGF, IGF-I, Acidic FGF, Basic FGF, Vasopressin

The "14-Ser peptide" AQPYPQGNHEASYG (SEQ ID NO: 15) increased cellnumber by 25-30% compared to control at four days. EGF (50 ng/ml) alonestimulated growth by 31% in the absence of "the 14-Ser peptide," and by61% in its presence, indicating that the two mitogens were additive.

IGF-I (50 ng/ml) stimulated growth by 20%; it was less potent than the14-Ser peptide. When both were added a 38% stimulation was observed.Although less than the expected 45% if fully additive, the mitogeniceffect of IGF-I appeared additive to that of the 14-Ser peptide.

Acidic FGF (100 pg/ml), basic FGF (1,000 pg/ml), and vasopressin (75pg/ml) stimulated growth by 17%, 18%, and 14%, respectively, compared tothe 14-Ser peptide which increased cell number by 25%. Each of theseknown peptide growth factors was additive with the 14-Ser peptidepeptide; acidic FGF by 44%; basic FGF, 41%; and vasopressin, 36%.

In summary, the 14-Ser peptide WGF-derived peptide is a potent mitogenbecause it is equivalent to, or more potent than, each of five knownpeptide growth factors for kidney epithelial cells. In addition, thegrowth-promoting effect of the 14-Ser peptide was additive with each ofthe five growth factors suggesting that it acts by a different receptorand/or signal transduction pathway than they do. Finally, the additivemitogenic effects suggest that the 14-Ser peptide, in combination withone or more of the known growth factors, could act in vivo to speedrepair and regeneration of the injured kidney during acute renalfailure.

2. High Potassium Medium, High Sodium Medium

Raising the potassium concentration from the control value of 5.4 mM to10.4 mM, or the sodium concentration form 155 mM (control) to 180 mM byaddition of the appropriate salt solution serves as a mitogenic signalfor kidney epithelial cells.

When the potassium concentration of the culture medium was raised from5.4 to 10.4 mM by adding a solution of potassium chloride, the cellnumber was increased by 18%. Addition of the 14-Ser peptide was expectedto enhance growth by 43%; an increase of 35% was observed.

Raising the sodium concentration of the medium from 155 mM to 180 mM byaddition of sodium chloride solution increased growth by 25%. When the14-Ser peptide was added an increment in growth of 41% was observed,less than the predicted 50%.

In summary, each of the two ionic mitbgenic signals were additive withthe 14-Ser peptide mitogen.

3. Time Required for the Growth Enhancing Effect of 14-Ser

To gain additional insight into the affinity of the 14-Ser WGF-derivedpeptide for the cell surface, experiments were performed to determinethe minimum amount of time required for the ligand to be in contact withrenal cells to irreversibly commit them to accelerated growth.

Confluent monolayers of BSC-1 cells are prepared as herein and exposedto the 14-Ser peptide (0.5 μg/ml) for different amounts of time (0 to 30minutes). At the end of the defined exposure period, culture mediumcontaining the mitogen was aspirated, the monolayer was rinsed to removenonadherent peptide, and fresh medium was added. Four days later thenumber of cells in the culture was counted.

Exposure of cells to the 14-Ser peptide for 2 minutes was sufficient tocommit them to maximal growth stimulation (29%); half-maximalstimulation was at 1 minute. The 40-amino acid peptide (see Table 2)behaved similarly. In contrast, the commitment time for EGF is 4minutes, and for vasopressin it is 2 minutes. Thus the 14-Ser peptideapparently requires a remarkably short time (2 minutes) to irreversiblybind to the surface receptor and commit cells to accelerated growth. Thetime required is equivalent to or less than other known peptide mitogens(EGF, 4 minutes; vasopressin, 2 minutes; low sodium growth factor, 5minutes; high potassium medium, 6 minutes; adenosine monophosphate, 14hours).

Purification of WGF

A protocol to prepare one liter of WGF conditioned medium is describedherein. The usual yield of WGF protein is about 50 ng per liter asestimated by SDS-polyacrylamide electrophoresis and amino acidcompositional analysis.

Conditioned Media Preparation: 100 cultures of BSC-1 cells are grown toconfluence (6-8×10⁶ cells) on plastic tissue culture dishes (Nunc)having a diameter of 10 cm in Dulbecco's modified Eagle's mediumcontaining 2% calf serum. The medium is aspirated, and the culture isrinsed with phosphate-buffered saline (PBS) (Sigma) solution to removemedium and serum. Then 10 ml of PBS is added to each dish in preparationfor wounding. A total of 5 wounds to the cell monolayer are rapidly madeby scraping a 200 μL plastic pipet tip across the surface of the dishfrom edge to edge. About ten minutes later (9.5 minutes is preferred)the conditioned buffer is decanted into a plastic Nalgene beaker.

Isolation and Purification of WGF: After conditioning, pooledconditioned buffer is sterile-filtered (CoStar, 0.2 μm pore size) toremove any debris or detached cells into silanized (Aquasil, Pierce)glass bottles (Wheaton). The sterile conditioned buffer is diafiltered,desalted, and concentrated using a YM-30 disc membrane (Amicon) at 4° C.The concentrated material is then sterile filtered (Millex HA, 0.45 Fm)at room temperature, loaded onto a 1 ml heparin-affinity cartridge andeluted with a solution of 1 M NaCl. The cartridge is first exposed to0.4 M NaCl in 10 mM sodium phosphate (pH 7.4) to elute nonmitogenicmaterial; the same buffer containing 1 M NaCl is then used to elutemitogenic activity from the cartridge. The eluate is desalted, and thevolume is reduced using a Centricon 3 filter (Amicon) by centrifugation(7500 g for 2 hours at 4° C.). The concentrate (300-400 μl) is loadedonto a C₄ -reversed phase HPLC column (250 mm×4.6 mm, particle size 5μm) (Vydac), and is eluted with a gradient of acetonitrile (1-80%) intrifluoroacetic acid (0.1%) (TFA) for 50 minutes using a Beckman GoldChromatographic System. At least 5 different protein peaks (monitored byabsorbance at 214 nm) that exhibit mitogenic activity can be readilyidentified. However, the peak that elutes at ˜55% acetonitrile routinelyexhibits the most mitogenic activity and reproducibility in differentisolates. This eluate is collected by hand into a silanized Eppendorftube, loaded onto a C₈ column (Vydac), and rechromatographed using thesame acetonitrile/TFA gradient described above. Again the mitogenicactivity elutes at ˜55% acetonitrile. Growth-promoting activity andtotal protein content is monitored at each step during the purificationprocess. Depending upon the number of liters of conditioned bufferprocessed, it may be necessary to run a second C₈ column to optimizeseparation of the peak of interest. This bioactive material is thenreduced in volume using a vacuum concentrator (Savant), and subsequentlyloaded onto a 12.5% SDS gel for electrophoretic separation.

The M_(r) of WGF is estimated by comparison of its electrophoreticmigration to that of standard proteins of known molecular size.Different proteins in the gel are visualized by staining with silver(BioRad), or after blotting onto a PVDF membrane (Millipore) andstaining the blot with Coomassie blue dye (Gibco/BRL). More than oneband usually appears. To determine which band represented the mitogenicform of WGF, an unstained nonreducing gel was sliced into 2-mm widefragments after electrophoresis, and each was eluted for 18 hours withagitation at room temperature in PBS containing acetonitrile (3%) andbovine serum albumin (0.1%). The eluate of each fragment was added to aculture of BSC-1 cells to determine its capacity to stimulate cellgrowth. This experimental strategy revealed that the active WGF proteinshad a M_(r) of 22 and 45 kDa.

WGF appears to be released from cells after wounding; it does not appearin the culture medium of nonwounded cultures. Evidence in support ofthis conclusion was obtained by the following experiment. Conditionedbuffer (1550 ml) from scrape-wounded cultures was obtained as describedabove and resulted in a C₈ -HPLC peak that eluted at 55% acetonitrileand exhibited growth-promoting activity, whereas the same volume (1550ml) of buffer exposed to nonwounded cultures subjected to the samepurification protocol did not display this protein peak or mitogenicactivity.

NH₂ -Terminal Sequence of Wound Growth Factor

To obtain the amino acid sequence of the NH₂ -terminus of WGF, 2.5 μg ofC₈ -purified protein was obtained and subjected to electrophoresis on a12.5% SDS-polyacrylamide gel. The relative electrophoretic mobility ofthe purified protein was compared to that of standard proteins of knownmolecular size. After blotting onto a PVDF membrane (Millipore) andstaining with Coomassie Blue dye, two bands were seen corresponding tosizes of 45 kDa and 22 kDa. They were subsequently cut out of the blotand then loaded onto an ABI microsequencer. The results ofmicrosequencing are expressed using conventional abbreviations andsymbols for the amino acids listed below:

    ______________________________________                                        A, Ala, alanine                                                                           C, Cys, cysteine                                                                            D, Asp, Aspartic acid                               E, Glu, glutamic acid                                                                     F, Phe, phenylalanine                                                                       G, Gly, glycine                                     H, His, histidine                                                                         I, Ile, isoleucine                                                                          K, Lys, lysine                                      L, Leu, leucine                                                                           M, Met, methionine                                                                          N, Asn, asparagine                                  P, Pro, proline                                                                           Q, Gln, glutamine                                                                           R, Arg, arginine                                    S, Ser, serine                                                                            T, Thr, threonine                                                                           V, Val, valine                                      W, Trp, tryptophan                                                                        Y, Tyr, tyrosine                                                                            M--NH.sub.2, methionine                             X, identity not           amide                                               determined                pE, pyroglutamic acid                               ______________________________________                                    

A total of four determinations of the amino-terminal sequence of WGFhave been made on different batches of conditioned media. The firstfourteen amino acids of the 45 kDa protein in the amino- tocarboxy-orientation (NH₂ →COOH) are, wherein the numbers refer topositions using the first amino acid as number one:

    ______________________________________                                        1           5        10    12  14                                             NH.sub.2 - A Q P Y P Q G N H E X A/S Y G--COOH (SEQ                           ID NO: 1)                                                                     ______________________________________                                    

Determination of the sequence of three different isolates of the 22 kDaprotein are:

    ______________________________________                                        1            5        10       16                                             NH.sub.2 - A Q P Y P Q G N H E A T S S S F--COOH                              (SEQ ID NO: 4)                                                                NH.sub.2 - A Q P Y P Q G N H E A T S S/Y - COOH                               (SEQ ID NO: 32)                                                               NH.sub.2 - A Q P Y P Q G N H E A T - COOH (SEQ ID                             NO: 7)                                                                        ______________________________________                                    

The longest is 16 amino acids.

Importantly, the first 10 amino acids are identical in each of the foursequence determinations, suggesting that the 22 kDa protein could be afragment or breakdown product of the 45 kDa protein. However, thedifferent sequences of amino acids 12 to 14 in the 22 and 45 kDaproteins suggest that they could represent two WGF isoforms.

The NH₂ -Terminal Domain of WGF is Mitogenic For Kidney Epithelial Cells

A sequence of the eleven NH₂ -terminal amino acids AQPYPQGNHEA (11-mer)(SEQ ID NO: 6) of the 22 kDa protein was used to prepare a syntheticpeptide linked to a branched lysine core. This multiple antigenicpeptide system (MAPS) was used to immunize animals to prepare apolyclonal antibody that would recognize WGF. The MAPS protein has apolylysine backbone that is attached to a resin at one end and has fourbranches at the other; each branch is ligated to one molecule of the11-mer peptide. Surprisingly, when tested for growth-promoting activity,the MAPS peptide stimulated DNA synthesis and proliferation of monkeykidney epithelial cells of the BSC-1 line. Its maximal growth-promotingeffect was similar to that of native WGF. When compared to a MAPSprotein prepared from a different 11 amino acid sequence and another oneprepared using an unrelated 16 amino acid sequence, only the sequencebased on WGF protein exhibited mitogenic activity. In subsequentexperiments, it was shown that the 11-amino acid peptide, in the absenceof the branched lysine core, stimulated proliferation of these kidneycells to the same extent as did purified WGF protein. Based on two celltypes tested, the 11-mer WGF peptide stimulated mitogenic activity ofrenal epithelial cells but did not stimulate growth of murine 3T3fibroblasts.

In addition, the growth-promoting effect of the 11-mer peptide and ofwound conditioned buffer were not additive, suggesting that the 11-merpeptide stimulates cell proliferation by the same receptor and signalingpathway as does intact WGF. These unexpected results suggest that the 11amino acids of the NH₂ -terminus represent a mitogenic domain of WGF,although other growth-promoting domains may be contained in the nativeprotein whose estimated length is about 400 amino acids.

FIG. 1 shows equivalent mitogenic dose responses of a synthetic 11-merpeptide, AQPYPQGNHEA (SEQ ID NO: 6) which represents native (22 kDa)amino-terminal WGF, and the most active hexapeptide synthesized of thepresent invention YPEGNH (SEQ ID NO: 33), which differs from native WGFby replacement of a glutamine (Q) (charge=0) residue with glutamic acid(E) (charge=-1). However, the native sequence is preferred (see Table2). The "native" sequence is that found in the 22 or 45 kDa WGF asobtained from cultured cells. The peptides are purified byreversed-phase HPLC on a C₁₈ column using a gradient of acetonitrile(1-80%) in 0.1% trifluoroacetic acid, lyophilized, and then dissolved intissue culture medium.

Nontransformed monkey kidney epithelial cells of the BSC-1 line weregrown to confluence in Dulbecco's modified Eagle's medium containing 1%calf serum and 1.6 μM biotin at 38° C. in a CO₂ incubator. When cellsachieved a density of about 10⁶ per dish, the spent medium was removedand replaced with fresh medium containing 0.5% calf serum and differentamounts of the two peptides. Four days later the number of cells in eachculture was counted. Each value is the mean of 3 separate cultures.

Mitogenicity of WGF-Derived Peptides

To delineate the sequence of the smallest peptide that could stimulaterenal cell growth, synthetic peptides of different lengths were prepared(see below), and purified by reversed-phase HPLC on a C₁₈ column.Mitogenic activity was assessed in a culture of BSC-1 cells by countingthe number of cells after exposure to a specified concentration (0.5 to20 μg/ml) of peptide for four days, and comparing the result to growthin the absence of added peptide.

PEPTIDES

    ______________________________________                                                     1      5        10                                               WGF 11-mer:   NH.sub.2 -A Q P Y P Q G N H E A--COOH                                         (SEQ ID NO: 6)                                                  9-mer:        .sub.    Q P Y P Q G N H E                                                    (SEQ ID NO: 20)                                                 8-mer:        .sub.      P Y P Q G N H E                                                    (SEQ ID NO: 23)                                                 7-mer:        .sub.      P Y P Q G N H                                                      (SEQ ID NO: 25)                                                 7-mer:             Y P Q G N H E                                                            (SEQ ID NO: 26)                                                 6-mer:             Y P Q G N H                                                              (SEQ ID NO: 2)                                                  ______________________________________                                    

Like the 11-mer, each of five shorter peptides, (6 to 9 amino acidslong) stimulated renal cell growth maximally to the same extent (25-30%)as did native WGF.

The following peptides (4 to 6 amino acids long) represent domains ofNH₂ -terminal WGF that did not stimulate cell growth, as compared to theactive 11-mer of line 1:

    __________________________________________________________________________                1    5    10                                                      WGF 11-mer:                                                                             NH.sub.2 -A Q P Y P Q G N H E A--COOH                                                            (SEQ ID NO: 6)                                   6-mer:    .sub.    Q P Y P Q G                                                                             (SEQ ID NO: 34)                                  4-mer:             G N H E   (SEQ ID NO: 35)                                  5-mer:          P Q G N H    (SEQ ID NO: 36)                                  5-mer:         Y P Q G N     (SEQ ID NO: 3)                                   __________________________________________________________________________

The following 6-mer and 7-mer peptides whose sequences/differ from WGFalso did not stimulate cell growth:

    ______________________________________                                        6-mer:      Y P R G N H    (SEQ ID NO: 37)                                    7-mer:      L K Y S G Q D  (SEQ ID NO: 38)                                    ______________________________________                                    

The results presented above indicate that maximal mitogenic activity ofpeptides based on the native sequence resides in a 6-mer whose sequenceis YPQGNH (SEQ ID NO: 2) because:

(1) peptides containing this sequence that are 7, 8, 9 or 11 amino acidslong stimulate growth to the same extent,

(2) each of two 5-mers that lack one amino acid from either the NH₂ - or--COOH terminus of the 6-mer are not mitogenic to the same extent,

(3) a 6-mer (YPRGNH) (SEQ ID NO: 37) that differs from YPQGNH (SEQ IDNO: 2) in only a single amino acid is not mitogenic to the same extent,

(4) another 6-mer (QPYPQG) (SEQ ID NO: 34) that differs from YPQGNH (SEQID NO: 2) in two amino acids is not mitogenic, and

(5) neither a 4-mer (GNHE) (SEQ ID NO: 35) whose sequence is found inWGF, nor a 7-mer (LKYSGQD) (SEQ ID NO: 38) having an unrelated sequence,is mitogenic.

Thus the NH₂ -terminus of native WGF contains an hexapeptide sequence,NH₂ -tyrosine-proline-glutamine-glycine-asparagine-histidine-COOH (SEQID NO: 2), that is mitogenic for renal epithelial cells, as exemplifiedby the BSC-1 line.

Peptides of various lengths are also within the scope of the presentinvention, as long as the mitogenic hexamer sequence is included.Peptides of various lengths may be used individually or combined invarious ways for specific purposes. These possibilities include (NH₂-terminus→COOH terminus):

AQPYPQGNHEATSSSF (SEQ ID NO: 4)

AQPYPQGNHEATSSS (SEQ ID NO: 5)

AQPYPQGNHEATSS (SEQ ID NO: 9)

AQPYPQGNHEATS (SEQ ID NO: 8)

AQPYPQGNHEAT (SEQ ID NO: 7)

YPQGNHEATSSSF (SEQ ID NO: 27)

YPQGNHEATSSS (SEQ ID NO: 28)

YPQGNHEATSS (SEQ ID NO: 29)

YPQGNHEATS (SEQ ID NO: 30)

YPQGNHEAT (SEQ ID NO: 31)

AQPYPQGNHEA (SEQ ID NO: 6)

AQPYPQGNHE (SEQ ID NO: 14)

QPYPQGNHEA (SEQ ID NO: 18)

AQPYPQGNH (SEQ ID NO: 19)

QPYPQGNHE (SEQ ID NO: 20)

PYPQGNHEA (SEQ ID NO: 21)

QPYPQGNH (SEQ ID NO: 22)

PYPQGNHE (SEQ ID NO: 23)

YPQGNHEA (SEQ ID NO: 24)

PYPQGNH (SEQ ID NO: 25)

YPQGNHE (SEQ ID NO: 26)

These peptides may be combined with other amino acid sequences at eitherend or both ends (NH₂ and COOH)

Characterization of Native and Modified WGF-Derived Amino-TerminalPeptides

Amino acid replacements in the amino-terminus of native WGF wereidentified that could alter peptide mitogenic activity. The objective ofthis effort was to maximize the growth-promoting activity of the peptidefor use in studies of its efficacy in treatment of acute renal failurein animal models of the syndrome. The strategy was to design and thenchemically synthesize diverse peptides by substituting structurallyrelated amino acids for those found in the native WGF sequence. Thepeptide of interest was then purified on a reversed-phase C₁₈ columnHPLC as described herein using a gradient of 1-80% acetonitrile in 0.1%trifluoroacetic acid (TFA). The purified peptide was then lyophilized toremove the acetonitrile and TFA and the lyophilized powder was weighed,dissolved in buffer and added to the culture medium to measure itseffect on the growth of monkey kidney epithelial cells as describedherein. To compare the relative potency of different peptides, onlythose molecules that stimulated growth maximally after 4 days in cultureby 25-30% were analyzed further by defining theirconcentration-dependence. A value termed the K_(1/2), was used. Thisterm is defined herein as the peptide concentration in micromoles (μM)at which the growth-stimulating effect is one-half the concentration atwhich maximal proliferation is observed. The lower the value, thegreater the mitogenic potency of the peptide.

                                      TABLE 2                                     __________________________________________________________________________    RELATIVE POTENCY OF WGF-DERIVED PEPTIDES                                      ON GROWTH OF KIDNEY EPITHELIAL CELLS                                                                          Growth-Stimulation                            Peptide Amino Acid Sequence     K.sub.1/2, μM                              __________________________________________________________________________    14-mer:                                                                           AQPYPQGNHEASYG (SEQ ID NO: 15)                                                                            0.126                                             AQPYPQGNHEASYG + keratan sulfate                                                                          0.067                                             AQPYPQGNHEASYG + heparin    0.040                                             AQPYPEGNHEASYG (SEQ ID NO: 39)                                                                            0.128                                             AQPYPQGNHEAAYG (SEQ ID NO: 11)                                                                            0.199                                             AQPYPQGNHEAAYG + keratan sulfate                                                                          0.054                                         11-mer:                                                                           YPQGNHEASYG (SEQ ID NO: 40) 0.166                                             YPQGNHEASYG + heparin       0.166                                             AQPYPQGNHEA (SEQ ID NO: 6)  0.260                                             AQPYPEGNHEA (SEQ ID NO: 41) 0.206                                         6-mers:                                                                           YPQGNH (SEQ ID NO: 2)       0.352                                             YPEGNH (SEQ ID NO: 33)      0.301                                             YPEGDH (SEQ ID NO: 42)      11.03                                             YPEGKH (SEQ ID NO: 43)      14.10                                             FPEGNH (SEQ ID NO: 44)      2.72                                              YPQGNH-amide                8.85                                          16-mer:                                                                           AQPYPQGNHEATSSSF (SEQ ID NO: 4)                                                                           0.218                                         40-mer:                                                                           AQPYPQGNHEAQPYPQGNHEAQPYPQGNHEAQPYPQGNHE                                                                  0.007                                             (SEQ ID NO: 49)                                                           __________________________________________________________________________

Amino acids designated by capitals are those present in native WGF,whereas the bold letters refer to conservative amino acid substitutions.

The results summarized in Table 2 indicate that it is longer peptides(40-mer>14-mer>11-mer>6-mer) containing the sequence YPQGNH (SEQ ID NO:2), that are the most potent. The longest non-repeating most potentpeptide tested is the 14-mer AQPYPQGNHEASYG (SEQ ID NO: 15), i.e., ithas the lowest K_(1/2), 0.126 μM. Note that exposure of the cells for 5minutes to the glycosaminoglycan, keratan sulfate (2 μg/ml) prior toaddition of the peptide, enhances the potency (K_(1/2) =0.067 μM) andheparin (1 μg/ml) is even more effective (0.040 μM). In rat studies the14-mer was also effective in increasing survival of rats in which ARFwas induced by mercuric chloride. The 14-mer with serine in position 12was more effective than the 11-mer with glutamic acid in position 6.

The length of the 14-mer AQPYPQGNHEASYG (SEQ ID NO: 15) provokedinterest about the three-dimensional structure of the peptide. Circulardichroism was carried out and revealed a spectrum consistent with abeta-pleated sheet conformation.

It is of interest that the potency of the 11-mer peptide AQPYPQGNHEA(SEQ ID NO: 6) (native WGF sequence) is similar to that of the synthetichexapeptide YPEGNH (SEQ ID NO: 33) (K_(1/2) ˜0.3 μM) which has a singlesubstitution: glutamic acid (E) replacing the native glutamine (Q).Several highly conservative modifications of the 6-mer sequence resultin drastic reductions in potency: replacement of the tyrosine (Y)residue with phenylalanine (F), substitution of asparagine (N) withaspartic acid (D) or lysine (K), or amidation of the carboxy-terminus.

Evidence to support the hypothesis that WGF and its peptides exert theirmitogenic effect via a cell surface receptor mechanism was obtained byshowing that pentameric peptides that lack one amino acid at eitherterminus of the hexapeptide YPQGNH (SEQ ID NO: 2), block theproliferative effect of intact hexamer. In these studies the pentamers,PQGNH (SEQ ID NO: 36) or YPQGN (SEQ ID NO: 3) were synthesized, purifiedby HPLC and then assayed for growth-promoting activity. Neither wasmitogenic. When either pentamer was added to the cell culture medium, itblocked the capacity of the hexamer YPQGNH (SEQ ID NO: 2) added at thesame time or afterwards to stimulate cell growth. These observationssuggest that the pentamers and the hexapeptide compete for the samebinding site/receptor on the plasma membrane. Because the pentamers arenot mitogenic, when they bind to the site, growth is not observed. Afterpentamers are bound to the site, the pentamers appear to prevent thehexapeptide from gaining access to the membrane.

Additional evidence that the peptides have a high potency for renalepithelial cells was obtained by defining the time required forinteraction between peptide and cells for irreversible commitment toaccelerated proliferation. In these experiments a solution containingthe peptide of interest was added to the cell monolayer; culture mediumwas then aspirated at a specified time thereafter. Then fresh mediumwithout the peptide was added and the number of cells was counted fourdays later. Contact of the cells with the peptide YPQGNH (SEQ ID NO: 2)for 2 minutes resulted in maximal growth-promoting activity, whereas thepeptide could be completely washed off the cells at earlier times (0.5,1, or 1.5 minutes) without stimulating growth. When the cells wereexposed to keratan sulfate (2 μg/ml) for 5 minutes before adding thepeptide, only 1 minute of exposure was necessary for the maximalmitogenic effect to be observed. Similar results were obtained when thepeptide YPEGNH (SEQ ID NO: 33) was tested; 3 minutes of exposure wassufficient to obtain a maximal growth response, but only 1.5 minuteswhen the cells were pretreated with keratan sulfate. Thus, keratansulfate enhances the mitogenic potency of not only native WGF proteinbut WGF-derived peptides as well.

A search of the seven protein sequence databases (Blaster) operated byNCBI revealed that the NH₂ -terminal sequence of WGF is novel and haslimited homology with two known mitogenic proteins, gastrin releasingpeptide (GRP) and bombesin, as shown below, using as the first positionthe first amino acid of the NH₂ terminus:

    ______________________________________                                                1     10       20     27                                              human GRP:                                                                              VPLPAGGGTVLTKMYPRGNHWAVGHLM--NH.sub.2                                         (SEQ ID NO: 45)                                                     porcine GRP:                                                                            APVSVGGGTVLAKMYPRGNHWAVGHLM--NH.sub.2                                         (SEQ ID NO: 46)                                                                1   5      10       16                                             WGF:    45 kDa   AQPYPQGNHEXAYG (SEQ ID NO: 1)                                                        S                                                             22 kDa   AQPYPQGNHEATSSSF                                                                             (SEQ ID NO: 4)                                bombesin:                                                                             pEQRLGNQWAVGHLM--NH.sub.2                                                                         (SEQ ID NO: 47)                                   1                10      14                                                   ______________________________________                                    

Symbols for amino acids in bold type represent identities betweendifferent proteins.

Alignment between the 14 amino acids of the NH₂ -terminus of WGF and thebombesin molecule is limited to the consecutive GN residues (amino acids#7,8 in the 14 amino acid active peptide of WGF; #5,6 in bombesin), andan A residue (amino acid #11 in WGF; #9 in bombesin). Previous reports(Broccardo, et al., 1975; and Heimbrook et al., 1988) indicate that theseven COOH-terminal amino acids of bombesin and of GRP are identical(WAVGHLM-amide) (SEQ ID NO: 48), and are also the locus of mitogenicactivity in these two proteins. A synthetic peptide comprising thissequence of amino acids is mitogenic for BSC-1 cells, but its K_(1/2) is˜10 μM. Importantly, the mitogenic sequence of these seven COOH-terminalamino acids is not found in the mitogenic 16 amino acids of the NH₂-terminus of WGF.

The functional differences between WGF and other known sequences includethat the hexapeptide YPQGNH (SEQ ID NO: 2) is mitogenic for renalepithelial cells, whereas the GRP-derived hexapeptide YPRGNH (SEQ ID NO:37) is at most borderline mitogenic (10% stimulation) for renalepithelial cells and is not reported to be necessary for mitogenicstimulation of fibroblasts.

It is of interest that the mitogenic hexapeptide derived from the NH₂-terminus of WGF, YPQGNH (SEQ ID NO: 2) (amino acids #4 to 9), differsin only a single amino acid (Q→R) from an hexapeptide sequence in humanand porcine GRP, YPRGNH (SEQ ID NO: 37) (amino acids #15 to 20), adomain that is not known to be mitogenic (Heimbrook et al., 1988). Thereis no prior teaching or suggestion to make an amino acid substitution atthis position, no suggestion to make the particular substitution that isin WGF, nor any such a suggestion that substitution would confermitogenic-stimulating activity on WGF.

These results indicate that the WGF-derived hexapeptide, YPQGNH (SEQ IDNO: 2), is a novel mitogen for renal epithelial cells.

Further Characterization of Mitogenic Peptides Derived From the NH₂-Terminus of Wound Growth Factor

As discussed in previous sections, purification of the Wound GrowthFactor (WGF) from conditioned medium of scrape-wounded monkey kidneyepithelial cells (BSC-1 line) revealed two mitogenic proteins; 22 kDaand 45 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Microsequencing of the NH₂ -termini of these proteinssuggested that they may be isoforms because the first 10 amino acids ofeach were identical. A total of 16 amino acids were identified at theNH₂ -terminus of the 22 kDa isoform, and 14 for the 45 kDa isoform.However, for the 45 kDa isoform, the amino acid at position 11 was notidentified, and at position 12, alanine or serine were equivalentdesignations.

    ______________________________________                                                 1            12        16                                            22 kDa isoform: NH.sub.2 --A Q P Y P Q G N H E A                                                    T      S S S F --COOH                                                       (SEQ ID NO: 4)                                                     1          10    12    14                                            45 kDa isoform: NH.sub.2 -- A Q P Y P Q G N H E X A/S Y G --COOH                                  (SEQ ID NO: 1)                                            ______________________________________                                    

Peptides of various lengths are within the scope of the presentinvention, as long as the mitogenic hexamer sequence, NH₂-tyrosine-proline-glutamine-glycine-asparagine-histidine-COOH (YPQGNH)(SEQ ID NO: 2) is included.

Amino acid replacements were identified in the amino-terminus of nativeWGF that could maximize the growth-promoting activity of the peptide foruse in studies of its efficacy in treatment of acute renal failure in arat model of this syndrome. The strategy was to design and thenchemically synthesize diverse peptides by substituting structurallyrelated amino acids for those found in the native WGF sequence.

The peptide of interest was then purified on a reversed-phase C₁₈ HPLCcolumn using a gradient of 1-80% acetonitrile in 0.1% trifluoroaceticacid (TFA). The purified peptide was then lyophilized to remove theacetonitrile and TFA and the lyophilized powder was weighed, dissolvedin buffer and added to the tissue culture medium to measure its effecton growth of monkey kidney epithelial cells.

To compare the relative potency of different peptides, only thosemolecules that stimulated growth maximally after 4 days in culture by25-30% were analyzed further by defining their concentration-dependence.A value termed the K_(1/2), was used. This term is defined herein as thepeptide concentration in micromoles (μM) at which the growth-stimulatingeffect is one-half the concentration at which maximal proliferation isobserved. The lower the value, the greater the mitogenic potency of thepeptide.

Table 2 indicates that the 14-amino acid peptide AQPYPQGNHEASYG (SEQ IDNO: 15) with a serine residue at position 12 (14-Ser) (K_(1/2) =0.126μM) is a more potent mitogen than AQPYPQGNHEAAYG (SEQ ID NO: 11) havingan alanine residue in that position (K_(1/2) =0.199 μM). When theglycosaminoglycan keratan sulfate was added at a concentration of 10μg/ml the mitogenic potency was enhanced because the K_(1/2) fell to0.067 μM. In addition, the glycosaminoglycan heparin, added at aconcentration of 1 μg/ml was even more effective, reducing the K_(1/2)to 0.040 μM. At the optimal concentrations employed in theseexperiments, a molecular ratio of 1 peptide molecule: 1glycosaminoglycan (GAG) molecule was added to the cells for eitherkeratan sulfate or heparin. The stimulatory effect of glycosaminoglycanswas mediated by formation of a peptide-GAG complex via ionic bonds(cationic histidine of peptide to anionic sulfate of either GAG) or byhydrogen bonding (hydroxyl groups of serine/tyrosine of peptide tooxygen residue of GAG carbohydrate). The peptide-GAG complex couldstabilize/facilitate binding of the peptide to its receptor on the cellsurface, as has been proposed to explain the enhanced mitogenic effectof acidic fibroblast growth factor (FGF-1) with heparin that has beenobserved with diverse types of cells. Replacement of a glutamine (Q)residue in position 6 with a glutamic acid residue (E) did not altermitogenic potency of 14-Ser peptide(K_(1/2) =0.128 μM), although thisreplacement strategy does reduce the K_(1/2) for the 6-amino acidpeptide YPQGNH (SEQ ID NO: 2) (K_(1/2) =0.352 to 0.301 μM), and for the11-amino acid peptide AQPYPQGNHEA (SEQ ID NO: 6) (K_(1/2) =0.260 to0.206 μM).

Shorter peptides of 11 amino acids in length such as AQPYPQGNHEA (SEQ IDNO: 6) (K_(1/2) =0.260 μM), and 6 amino acids, YPQGNH (SEQ ID NO: 2)(K_(1/2) =0.332 μM) each lack the SYG terminal amino acids of the14-amino acid peptide AQPYPQGNHEASYG (SEQ ID NO: 15) (K_(1/2) =0.126μM), and are less potent as mitogens. To determine if an 11 amino acidpeptide containing this SYG sequence was required for maximalmitogenesis, a peptide with the sequence YPQGNHEASYG (SEQ ID NO: 40) wassynthesized, purified, and then assayed for activity. Interestingly itsK_(1/2) was 0.166 μM in the absence or presence of heparin, indicatingthat the full 14 amino acid length was required for the maximalmitogenic effect.

A peptide synthesized using the longest known WGF sequence, the 16 aminoacids of the 22 kDa isoform, AQPYPQGNHEATSSSF (SEQ ID NO: 4), was alsotested for its mitogenic activity (K_(1/2) =0.218 μM). It was not aspotent as the 14-amino acid peptide AQPYPQGNHEASYG (SEQ ID NO: 15)derived from the NH₂ -terminus of the 45 kDa isoform.

Finally, a 40-amino acid peptide was synthesized. It is a tetramer ofthe first 10 amino acids which are identical in the 22 kDa and the 45kDa isoforms of WGF. This is the most potent WGF peptide yet identified,having a K_(1/2) =0.007 μM. The enhanced mitogenic potency is presumablyrelated to its relatively great length which could stabilize ligandbinding at the cell surface receptor site by ionic or hydrophobicinteractions, and/or hydrogen bonding. Ultimately, it may prove the mostefficacious WGF-derived peptide under clinical conditions because of itsgreat potency.

The 14-amino acid peptide (14-Ser), because of its high mitogenicpotency and relatively short length, was chosen for further studies incells, and for use in the treatment of rats with mercuricchloride-induced acute renal failure.

An Antibody to the Factor

The 11 amino acids of the NH₂ -terminus of WGF were used to prepare twodifferent conjugated peptide antigens. One was the MAPS proteindescribed above in which the 11-mer peptide was linked to a branchedpolylysine backbone, and the other was a conjugate in which the 11-merwas chemically joined to keyhole limpet hemocyanin (KLH). The MAPSprotein and KLH-conjugate were used to immunize rabbits. The antibodythat results from these strategies serves to detect native WGF proteinin urine, serum, and tissue, and could also be used to obtain a cDNAclone encoding WGF by immunoscreening a kidney epithelial cell cDNAlibrary in λgt11.

Lack of immunogenicity of the peptide would favor its clinical utilityin the treatment of renal diseases such as acute renal failure.

Product Relevance of the WGF-Derived Mitogenic Hexapeptide

The synthetic hexapeptide, like native WGF, can be used to treat acuterenal failure, chronic renal diseases, and renal cell cancer. Becausepreparation of an antibody to an hexapeptide may not be readilysuccessful because of its small size, a goat or rabbit antibody againstthe 11-mer or a larger peptide may be preferred to detect WGF in urine,serum, and tissue of patients with renal diseases.

Production of WGF by Recombinant Genetic Methods

An appropriate vector to express WGF from the nucleotide sequenceencoding a full-length clone depends in part upon whether glycosylationof the protein is required for each of its biological effects. If WGF isnot glycosylated, a bacterial expression system may prove suitable, butif carbohydrate is definitively detected and shown to be required forfull activity, then a mammalian expression system such as ChineseHamster Ovary (CHO) cells would be more appropriate.

Because the synthetic 14-mer peptide, indeed an hexamer of theamino-terminus is mitogenic, it appears that glycosylation of theprotein is not required for mitogenic activity. However, glycosylationcould prove to be an important determinant in modulating the turnover(half-life) of the protein in vivo. This is the case for erythropoietin,for example.

A nucleotide sequence encoding for at least the mitogenic hexamer isprepared, linked to suitable regulatory elements, and incorporated intoan expression vector. A host cell is transformed with the vector, andthe host is placed in conditions suitable for expression. Afterexpression of the WGF gene or partial nucleotide sequence in arecombinant host, the recombinant protein is isolated from the culturemedium by using an antibody-affinity column prepared by conjugating anantibody to WGF to an appropriate matrix. This preparative strategyshould yield large quantities of the recombinant protein.

Alternatively, and more simply, sufficient amounts of the NH₂ -terminal14-Ser, 11 amino acid peptide, the hexapeptide YPQGNH (SEQ ID NO: 2), orother peptides expressing full mitogenic potency, can be prepared byconventional peptide synthesis.

Detailed techniques to produce WGF by recombinant systems are known tothose of skill of art.

In Vitro and in Vivo Models

In vitro and in vivo models of acute renal failure are useful to studytwo major biological characteristics of wound repair: cell migration andproliferation. These models are suitable for analyzing the effects orthe peptides of the present invention.

i. In vitro model: scrape-wounding of monolayer cultures of renalepithelial cells to simulate injured renal tubular epithelium.

In this model, high-density, quiescent monkey kidney epithelialmonolayer cultures (BSC-1 line) are wounded by mechanically scrapingaway defined regions of the monolayer to simulate the effect of cellloss after tubular necrosis. The number of cells that migrate into thedenuded area is counted (Kartha and Toback, 1992). It was found thatcell migration is independent of cell proliferation, although bothprocesses can be studied in this experimental preparation.

The model is useful to study the kinetics of renal epithelial cellmigration, and identify genes whose expression is induced or repressedafter wounding. The biological characteristics and potency of the WGF issuitable for investigations using this system.

ii. In vivo model: mercuric chloride (nephrotoxic) and renal clampingartery (ischemic) models of acute renal failure (ARF) in the rat.

Previous studies using this model system have characterized theclinical, histological, biochemical, and functional correlates of theacute renal failure syndrome. It was demonstrated that infusion of amixture of essential and nonessential amino acids stimulates synthesisof phosphatidylcholine and protein in regenerating rat renal tissue andreduces the level of kidney dysfunction after intravenous administrationof mercuric chloride (Toback, 1980). These studies showed thatbiochemical and functional repair of the injured kidney are not optimalin untreated animals and that provision of exogenous amino acids withglucose could speed recovery. The capacity of the WGF to enhanceregeneration after acute renal failure is studied in this model.

It is important to define the onset of abnormal kidney function andstructure, its course, and subsequent recovery. In humans, as in rats,kidney function during ARF is monitored by measurements of theconcentration of serum creatinine and blood urea nitrogen (BUN) on adaily basis. These measurements provide estimates of glomerularfiltration in the kidney which is one of its major functions. Creatinineenters the serum from muscle where it is released constantly as aconsequence of the metabolic turnover of creatine phosphate, whereas theBUN is the end-product of total body protein turnover. Both moleculesmake their way into the blood and because of their small size areexcreted in the urine. Thus the extent to which they accumulate in theblood and are not excreted in ARF provides a guide to the extent ofkidney injury.

Treatment of Chronic Renal Diseases

The mechanisms that mediate the progression of renal disease in diabeticnephropathy, interstitial nephritis, and chronic glomerulonephritis areunknown. Relentless loss of renal function results in the need forchronic dialysis treatment at a cost of billions of dollars each year inthe United States because at present there is no therapeutic strategy toslow or reverse the process. Patients early in the course of renaldisease are suitable candidates for growth factor therapy because noother alternative is now available to treat this condition. Theobjective of administration of WGF is to speed recovery of injured renalepithelial cells along the nephron thereby repairing the damage mediatedby the disease process, preserving renal function, and forestalling theneed for dialysis.

Treatment of Renal Cell Cancer

Renal cell cancer is often a slow growing condition that results inwidespread metastases to distant sites in the body making successfultreatment difficult. Studies using radioactive WGF permit its receptoron renal cells to be identified and isolated; antibodies directedagainst the receptor are then prepared. These antibodies serve tolocalize receptors on specific types of kidney cells along the nephron.Armed with this information about the cell-type specificity of WGFreceptors, strategies to treat cancers of renal cell types of interestinclude WGF or a specific WGF peptide that exhibits binding to thereceptor and can thereby be used to deliver an anticancer agent to theproliferating cells of interest. Such anticancer agents comprise WGF ora specific WGF peptide or peptides conjugated to a toxin, cytolyticantibody, or a radioisotope. Although treatment of the primary renaltumor is likely best carried out by surgical extirpation of the organ,treatment of cancer metastases, especially those of small size, could beimportant targets of these novel chemotherapeutic agents that would beformulated using knowledge of WGF structure and function.

Delivery of WGF to Patients

Delivery to patients is generally by intravenous infusion similar tocancer chemotherapy deliveries or experimental treatment of smallmammals. Another route is that used for treatment of anemia witherythropoietin (EPO) in renal failure wherein EPO is delivered every fewdays by subcutaneous injection.

Diagnosis of Renal Cell Injury by Use of an Antibody to WGF

The antibody to native WGF is prepared as disclosed herein and used todetermine the concentration of the growth factor in urine and blood.Distribution of WGF in different types of renal cells along the nephronis determined. The concentration of the growth factor in either blood orurine or both could increase as a result of renal injury. If the growthfactor is excreted in the urine as is epidermal growth factor, it may bepossible to determine if renal injury leads to increased urinaryexcretion of the new factors. If so, enzyme-linked immunosorbent assay(ELISA) kits to detect WGF are used to rapidly diagnose early renalinjury prior to a fall in glomerular filtration rate which is notdetectable with conventional laboratory tests until more than 50% ofkidney function is lost. This is particularly valuable in patientsreceiving drugs with nephrotoxic potential during treatment of severeinfections or malignancies. An antibody to WGF is incorporated into adiagnostic ELISA kit designed to detect the appearance of growth factorin blood and urine of patients with renal injury or neoplasia.

Detection of Renal Cancer in Chronic Hemodialysis Patients

If renal adenomas, cystadenomas, or carcinomas that occur in remnantkidneys of patients undergoing chronic hemodialysis are found tooverexpress the growth factor and excrete it in the urine, an ELISA kitcould provide a new early detection system for these lesions. Such adiagnostic kit could also be used in asymptomatic but high-riskindividuals. Renal cancer is now difficult to detect early because ittends to be asymptomatic until the tumor has grown to significant sizeor has metastasized widely.

Manipulations of Growth Factor, Structure and Functions

Analogs of the growth factor are developed to maximize thegrowth-promoting effect of WGF by optimizing specific binding to renalepithelial cell receptors. Inhibitors that block the biological actionof each factor by binding to receptors on the cell surface are alsouseful. Development of a synthetic or recombinant product that isresistant to degradation would prolong pharmacological activity in vivo.

EXAMPLES

The following examples are provided for illustration, not limitation.

Example 1 14-Ser Peptide is Effective in the Treatment of NephrotoxicAcute Renal Failure

Nephrotoxic and ischemic acute renal failure (ARF) inducedexperimentally in rats have long been used to model this syndrome inhumans. To determine if the mitogenic effect of 14-Ser, a WGF-derivedNH₂ -terminal peptide, would prove efficacious as a therapeutic agent inanimals with ARF, acute tubular necrosis was induced by injecting asolution of mercuric chloride (in normal saline) subcutaneously to ratsat a dose of 2.25 mg per kilogram body weight. Male rats weighed 200-225gm at the start of the experiment. This dose was used becausepreliminary experiments indicated it resulted in survival of 25-50% ofthe animals 7 days later. Blood was obtained from the tail vein each dayand the concentration of creatinine in the serum was measured and usedas an index of renal function. An increase in serum creatinineconcentration signals a decline in renal function because the injuredkidney is unable to excrete endogenous creatinine in the urine so itaccumulates in the blood.

The synthetic peptide AQPYPQGNHEASYG (14-Ser) (SEQ ID NO: 15) (dissolvedin sterile 0.01% bovine serum albumin in phosphate-buffered saline) wasinjected subcutaneously into rats to determine its effect on survival,recovery of renal function, and stimulation of DNA synthesis. Multipledifferent peptides were studied including 14-Ser, AQPYPEGNHEASYG(14-mer) (SEQ ID NO: 39), AQPYPQGNHEATSSSF (16-mer) (SEQ ID NO: 4),AQPYPQGNHEA (11-mer) (SEQ ID NO: 6), and AQPYPEGNHEA (11-mer) (SEQ IDNO: 41). Different concentrations and times of administration weretested and compared. The results indicated that 14-Ser was the mosteffective WGF-derived peptide; it improved survival and recovery ofrenal function, and stimulated DNA synthesis in the regenerating kidneyafter mercuric chloride-induced ARF.

Acute tubular necrosis was induced in 44 rats by the subcutaneous (s.c.)injection of mercuric chloride, and a single dose of 14-Ser wasadministered s.c. 1 hour afterwards. Different amounts of the peptidewere given to assess its capacity to affect the outcome. Survival 7 dayslater was about twice as high in animals given 100-150 μg of peptide(63%) than in rats given 0-75 μg peptide (29%).

To determine the effect of the peptide on survival and recovery of renalfunction, acute tubular necrosis was induced in 59 rats; 29 received14-Ser (100 μg/rat) and 30 received an equal volume of the vehicle s.c.1 hour after administration of mercuric chloride. Enhanced survival (toppanel) and renal functional recovery (bottom panel) of rats given aWGF-derived peptide after mercuric chloride-induced acute tubularnecrosis are shown in FIG. 2A and FIG. 2B. Mercuric chloride wasinjected subcutaneously into each of the 59 rats, and 1 hour later 100μg of WGF peptide (14-Ser) (n=29 rats) or the vehicle (n=30) was given.Survival was greater in rats given the peptide than in animals receivingthe vehicle as early as day 3 after administration of mercuric chloride.Blood was obtained from the tail vein on the days indicated, and serumcreatinine concentration was measured. A similar increase in creatinineconcentration was observed in both groups of animals on day 1,signifying that they sustained the same extent of renal functionalinjury. On days 2, 3 and 4, the lower creatinine concentration inpeptide-treated rats indicates accelerated recovery of kidney function.

FIG. 2A indicates that the survival of rats given the peptide wassignificantly greater (chi squared, P=0.035) than those given vehicle 3days after the onset of the ARF syndrome, a difference that persistedduring the next 4 days. By day 7 only 20% of rats that received thevehicle were alive, whereas 48% of rats given the peptide survived (chisquared, P=0.012).

Assessment of renal function during the onset of the ARF syndromerevealed impaired function on day 1 when the creatinine concentrationincreased from the basal value of 0.5 mg/dL to 2.3 mg/dL. It was similarin animals treated with the peptide (n=29 rats) or the vehicle alone(n=30) (FIG. 2B), suggesting that the extent of renal injury wasequivalent in both peptide-treated and untreated animals. On day 2 theserum creatinine concentration was significantly lower inpeptide-treated rats (n=29) than in animals given the vehicle (n=30)(P=0.034, Student's t-test), indicating that the decline in renalfunction was less severe in rats given mercuric chloride followed by thepeptide. Evidence of a significantly more rapid recovery of renalfunction was also apparent on days 3 and 4 after the onset of ARF.Similar beneficial effect of peptide administration was observed whenthe blood urea nitrogen concentration was measured as an index of renalfunction. These results indicate that a WGF peptide that is mitogenicfor renal epithelial cells can speed renal functional recovery andenhance survival of rats with nephrotoxic ARF.

In a separate set of experiments, 14-Ser (n=7) or vehicle (n=9) wasgiven to rats 24 hours before mercuric chloride-induced injury. On day3, renal function assessed using the serum creatinine concentration wassignificantly better in rats given the peptide (P=0.039). Survival onday 4 was 71% in rats treated with the peptide but only 22% in thosegiven the vehicle (chi squared, P=0.049). Treatment with the 14-Serpeptide 24 hours before the mercuric chloride-induced insult appeared tohave a more favorable effect on survival than when given 1 hourafterwards, although the difference did not quite achieve statisticalsignificance.

To determine if the beneficial effects of 14-Ser peptide on survival andrenal function were the result of the mitogenic action of the peptide,DNA synthesis was measured in the kidneys of rats given mercuricchloride. 5-Bromo-2'-deoxyuridine (BrdU), a nonradioactive analog ofthymidine that is incorporated into DNA was used to label nuclei. Amonoclonal antibody to BrdU and diamidobenzidene staining were used todetect nuclei undergoing DNA synthesis in histological sections of renaltissue. BrdU was dissolved in 20% ethanol and was injectedintra-peritoneally 23 hours after administration of mercuric chloride.One hour later the kidneys were quickly perfused with a warm solution ofphosphate-buffered saline via an aortic catheter to eliminate red bloodcells from the organ and maintain patency of the renal tubules. Thecapsule was removed from the kidneys which were then bisectedlongitudinally, and fixed in formalin for 4 hours, and then in 70%ethanol. Tissue sections were prepared and BrdU-labeled nuclei weredetected as described above.

BrdU staining of kidneys of rats given no mercuric chloride revealedabout 3 labeled nuclei per high-power field (×400) in the subcapsularand aglomerular (inner) cortex. In the kidneys of a rats given mercuricchloride, extensive necrosis of tubular cells of the terminal portion(S₃) of the proximal nephron was observed, as expected in thiswell-characterized model system. The extent of necrosis was somewhatvariable from rat-to-rat but was always confined to the aglomerularcortex. Fewer labeled cells were seen in the aglomerular cortex in ratsgiven mercuric chloride 24 hours earlier than in cells of untreatedcontrol rats. Renal tissue from a rat given 14-Ser (100 μg) 24 hoursbefore administration of mercuric chloride was also examined. In thisanimal, 1 hour of BrdU labeling was carried out 24 hours after mercuricchloride as described herein. Microscopic examination revealed that manymore labeled nuclei were present adjacent to the zone of tubular cellnecrosis. This finding is important because it is in this boundary areaof the cortex where regenerating cells are expected to undergo mitosisand then migrate into nephrons in the necrotic zone to replace cellsthat were irreversibly injured by the nephrotoxic insult. Thus the14-Ser peptide appears to stimulate DNA synthesis in cells ofregenerating nephrons after mercuric chloride-induced tubular necrosis,and could thereby speed recovery of renal function and improve survivalafter ARF.

Example 2 Improved Survival with 11-mer WGF-Derived Peptide Treatment ofExperimental Acute Renal Failure

Experimental ARF was induced in rats by subcutaneous injection ofmercuric chloride. The synthetic peptide AQPYPEGNHEA (SEQ ID NO: 41)(dissolved in sterile 0.01% bovine serum albumin and PBS) was injectedsubcutaneously into rats two hours after mercuric chloride was given todemonstrate its beneficial effect on survival from the ARF syndrome. Inone experiment a total of 17 rats were injected with a dose of mercuricchloride shown to induce ARF. Then different amounts of the peptide(known to stimulate growth of kidney cells in culture) were injectedinto the animals to assess capacity to improve the outcome. Three dayslater, five of five animals given saline alone and three of three ratsgiven 50 micrograms of the peptide were dead. In contrast, two of threerats given 100 micrograms of peptide were alive, as were five of sixgiven 200 micrograms. In summary, none of eight rats (0%) given up to 50micrograms of peptide survived, whereas seven of nine (78%) given100-200 micrograms did. Thus, the 11-mer WGF peptide improves survivalin nephrotoxic ARF.

Example 3 Effect of Specific WGF-Derived Peptides on Kidney Repair

In addition, administration of specific WGF-derived peptides or thenative protein speeds repair of renal structure. Standard assays inrenal growth physiology and pathology are used to demonstrate that thegrowth-promoting (mitogenic) effect of a WGF peptide or the 22 or 45 kDaprotein stimulates an increased number of kidney cells to initiatesynthesis of DNA in preparation for cell division, as has been shown intissue culture.

Initially, sections of kidney tissue from rats with mercuricchloride-induced ARF are prepared and inspected under light microscopyto compare the extent of renal injury and repair in animals thatreceived WGF peptide and those that received a saline vehicle alone.Another measure of the effect of WGF on recovery is an histopathologicalassessment based on detailed microscopic inspection of kidney tissue. Ascoring system that grades the characteristic features of ARF andrecovery is used to assess and compare kidney tissue from rats that didor did not receive treatment with WGF. (Miller et al., 1994)

Depending upon methodologic considerations, radioactive thymidine can beused to measure the capacity of WGF protein or peptide to stimulaterenal DNA synthesis after ARF. In these experiments radioactivethymidine is injected intraperitoneally 1 hour before death, and itsincorporation into renal DNA is determined by subsequently extractingDNA from the tissue, and then measuring the amount of DNA by a chemicalassay, and its radioactivity by scintillation counting. Autoradiogramscan also be prepared and used to determine which cells in kidney tissuehave incorporated radioactive thymidine into DNA. All the techniquesreferred to are known in the art.

An ischemic model of ARF is also suitable. In these rats, pentobarbitalanesthesia is induced and the kidneys are deprived of blood flow for 60minutes by placing surgical clamps on each renal pedicle (artery, vein,ureter) that is first exposed at surgery. This is an established modelof ARF. After the clamps are released to restore blood flow to thekidneys, the surgical incision sites in the skin and muscle are closedwith skin clips, and the animals are treated with WGF peptide byinjection s.c. Measurements of recovery of renal function and structureare the same as for the toxic (mercuric chloride) model describedherein.

Example 4 Inhibition of WGF-Derived Peptide Mitogenesis by TGF-β2 andYPQGN (SEQ ID NO: 3) Peptide

To better understand the role of the 14-Ser peptide in renal cell growthregulation, agents that could inhibit its mitogenic effect were studied.

Transforming growth factor (TGF)-β2 is an autocrine growth inhibitorsecreted by BSC-1 cells that limits proliferation in culture. ExogenousTGF-β2 was added to the culture medium to determine its capacity toinhibit the growth-promoting effect of 14-Ser. TGF-β2 at a concentrationof 1 ng/ml was sufficient to abolish the 25% stimulation of growthinduced by 14-Ser. At a TGF-β2 concentration of 2 ng/ml, growth wasinhibited by 30% in the presence or absence of 14-Ser; at 10 ng/ml,inhibition was 60%. At a TGF-β2 concentration of 6 ng/ml, exogenous14-Ser up to 5 μg/ml did not reverse growth inhibition.

Because TGF-β2 inhibits proliferation induced by diverse mitogens, amore specific inhibitor was sought. Previously, studies of the 5-aminoacid WGF-derived peptides YPQGN (SEQ ID NO: 3) and PQGNH (SEQ ID NO: 36)were shown to inhibit the mitogenic effect of the 6-amino acid peptide,YPQGNH (SEQ ID NO: 2). These pentapeptides do not alter growth whenadded to cells. Cells were preincubated for 30 minutes with thepentapeptide YPQGN (SEQ ID NO: 3). The culture medium was thenaspirated, the monolayer was rinsed, fresh medium was added, and thenumber of cells counted 4 days later. Preincubation of cells with YPQGN(SEQ ID NO: 3) inhibited the mitogenic effect of the 6-amino acidpeptide. In addition, preincubation with YPQGN (SEQ ID NO: 3) for 10 or30 minutes completely blocked the growth-promoting effect of the 14-Serpeptide as well. Importantly, the mitogenic effect of partially-purifiedWound Growth Factor (passed over an heparin-affinity cartridge, not anHPLC column) was also completely abolished.

These observations suggest that the 14-Ser and YPQGNH (SEQ ID NO: 2)(WGF-derived) peptides, and partially-purified WGF bind to the same cellsurface receptor, which also has a high affinity for the pentapeptideYPQGN (SEQ ID NO: 3). The WGF receptor may be isolated and characterizedusing high affinity ligands such as the 14-Ser peptide and the 40-aminoacid peptide which can readily be radioiodinated for use as probes.

Example 5 Comparison Between the 14-Ser WGF-Derived Peptide and EGF onSurvival After Acute Renal Failure

The efficacy of the 14-Ser peptide as a novel therapeutic agent inmercuric chloride-induced ARF was compared to EGF, which was the firstpeptide growth factor shown to be effective in this animal model system.

Eight rats in each of three groups (n=24) were given either 14-Ser (100μg, s.c.) 1 hour after mercuric chloride (2.25 mg/kg s.c.), EGF (20 μg,s.c.) 2 hours after the toxin, or the vehicle (s.c.), and survival wasmonitored. Four days later, 63% of rats given the 14-Ser peptide werealive compared to 25% of animals receiving the vehicle (chi squared,P=0.0499); 50% of rats receiving EGF were alive (chi squared, P=N.S.).On day 5, 50% of rats given EGF were alive, compared to 12.5% of animalsgiven the vehicle (chi squared, P=0.0436). On day 6, survival wasidentical in rats given the 14-Ser peptide or EGF (37.5%) compared to12.% of animals treated with the vehicle. These observations indicatethat the 14-Ser peptide and EGF given after renal injury are similar intheir ability to improve survival from the ARF syndrome.

In a separate experiment, rats were given EGF (20 μg, s.c.) 24 hoursprior to administration of mercuric chloride to compare the effect ofthis growth factor to the 14-Ser peptide. Surprisingly, EGF treatmentwas not different than the vehicle, whereas the 14-Ser peptide is highlyprotective under these conditions. Thus, on day 4, survival of ratsgiven EGF was 25%, for vehicle, 31%, and for the 14-Ser WGF-derivedpeptide, 73%.

These studies indicate that the 14-Ser peptide is more effective thanEGF in promoting survival when administered prophylactically, i.e.,before the onset of renal injury. Administration of WGF-derived peptidecould prove particularly efficacious: (1) prior to surgical proceduresin patients with a high risk of developing ARF (e.g., the elderly,neonates), (2) in patients given potentially nephrotoxic agents such asantibiotics for treatment of sepsis or antineoplastic agents, and (3)for perfusion of donor kidneys prior to their transplantation into newhosts.

DOCUMENTS CITED

Broccardo, M., Falconieri Erspamer G., Melchiorri P., Negri L., and DeCastiglione, R., "Relative potency of bombesin-like peptides," Journalof Pharmacology 55:221-227 (1975).

Coimbra T. M., Cieslinski, D. A., and Humes, H. D. "Epidermal growthfactor accelerates renal repair in mercuric chloride nephrotoxicity,"American Journal of Physiology 259:F438-F443, 1990.

Heimbrook, D. C., Boyer, M. E., Garsky, V. M., Balishin, N. L., Kiefer,D. M., Oliff, A., and Riemen, M. W., "Minimal ligand analysis of gastrinreleasing peptide. Receptor binding and mitogenesis," Journal ofBiological Chemistry 263:7016-7019 (1988).

Kartha, S., and Toback, F. G., "Adenine nucleotides stimulate migrationin wounded cultures of kidney epithelial cells," Journal of ClinicalInvestigation, 90:288-292 (1992).

Kartha, S., and Toback, F. G. "Purine nucleotides stimulate DNAsynthesis in kidney epithelial cells in culture," American Journal ofPhysiology 249:F967-F972, 1985.

Levy, E. M., Viscoli, C. M. and Horowitz, R. I. The Effect of AcuteRenal Failure on Mortality, "A Cohort Analysis" Journal of the AmericanMedical Association 275:1489-1494 (1996).

Mendley, S. R., and Toback, F. G., "Autocrine and paracrine regulationof kidney epithelial cell growth," Annual Review of Physiology, 51:33-50(1989).

Miller, S. B., Martin, D. R., Kissane, J., and Hammerman, M. R."Hepatocyte growth factor accelerates recovery from acute ischemic renalinjury in rats." American Journal of Physiology 266:F129-F134 (1994).

Mordan, L. J., and Toback, "Growth of kidney epithelial cells inculture: Evidence for autocrine control," American Journal ofPhysiology, 246:C351-C354 (1984).

National Center for Health Statistics, National Institutes of Health,1995.

National Kidney and Urological Advisory Board, 1990 Long Range Plan.

Toback F. G., "Amino acid treatment of acute renal failure," InContemporary Issues in Nephrology, ed. Brenner, B. M. and Stein, J. H.Vol. 6, pp. 202-228, Churchill Livingstone, New York (1980).

Toback, F. G., "Control of renal regeneration after acute tubularnecrosis," Nephrology, Proceedings IXth International Congress ofNephrology, I:748-762 (1984).

Toback, F. G. "Induction of growth in kidney cells in culture by Na⁺,"Proceedings of the National Academy of Sciences, USA," 77:6654-6656,1980.

Toback, F. G., "Regeneration after acute tubular necrosis," KidneyInternational, 41:226-246 (1992).

Toback, F. G., Ekelman, K. B., and Ordonez, N. G. "Stimulation of DNAsynthesis in kidney epithelial cells in culture by potassium," AmericanJournal of Physiology 247: C14-C19, 1984.

Toback, et al., U.S. Pat. No. 5,135,856, issued Aug. 4, 1992 (b) andU.S. Pat. No. 5,476,922, issued Dec. 19, 1995.

Walsh-Reitz, M. M., Gluck, S. L., Waack, S. and Toback, F. G., "Loweringextracellular Na⁺ concentration releases autocrine growth factors fromrenal epithelial cells," Proceedings of the National Academy ofSciences, USA, 83:4764-4768 (1986).

Walsh-Reitz, M. M, and Toback, F. G. "Vasopressin stimulates growth ofrenal epithelial cells in culture," American Journal of Physiology 245:C365-370, 1983.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 49                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/product= "Unknown"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/product= "Ala or Ser"ORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Xa - #a Xaa Tyr Gly                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Tyr Pro Gln Gly Asn His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Tyr Pro Gln Gly Asn                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser Ser Ser Phe         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser Ser Ser             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser Ser                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser Tyr                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ala Tyr Gly                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ala Tyr                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ala                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ser Tyr Gly                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ser Tyr                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Ser                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Gln Pro Tyr Pro Gln Gly Asn His Glu Ala                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Gln Pro Tyr Pro Gln Gly Asn His Glu                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Pro Tyr Pro Gln Gly Asn His Glu Ala                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Gln Pro Tyr Pro Gln Gly Asn His                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Pro Tyr Pro Gln Gly Asn His Glu                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Tyr Pro Gln Gly Asn His Glu Ala                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                           - Pro Tyr Pro Gln Gly Asn His                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Tyr Pro Gln Gly Asn His Glu                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Tyr Pro Gln Gly Asn His Glu Ala Thr Ser Se - #r Ser Phe                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Tyr Pro Gln Gly Asn His Glu Ala Thr Ser Se - #r Ser                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Tyr Pro Gln Gly Asn His Glu Ala Thr Ser Se - #r                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Tyr Pro Gln Gly Asn His Glu Ala Thr Ser                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Tyr Pro Gln Gly Asn His Glu Ala Thr                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/product= "Ser or Tyr"ORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Thr Ser Xaa                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Tyr Pro Glu Gly Asn His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Gln Pro Tyr Pro Gln Gly                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Gly Asn His Glu                                                             - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Pro Gln Gly Asn His                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Tyr Pro Arg Gly Asn His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - Leu Lys Tyr Ser Gly Gln Asp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - Ala Gln Pro Tyr Pro Glu Gly Asn His Glu Al - #a Ser Tyr Gly                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - Tyr Pro Gln Gly Asn His Glu Ala Ser Tyr Gl - #y                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - Ala Gln Pro Tyr Pro Glu Gly Asn His Glu Al - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                - Tyr Pro Glu Gly Asp His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                - Tyr Pro Glu Gly Lys His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - Phe Pro Glu Gly Asn His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 27 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Val Pro Leu Pro Ala Gly Gly Gly Thr Val Le - #u Thr Lys Met Tyr Pro         #                15                                                           - Arg Gly Asn His Trp Ala Val Gly His Leu Me - #t                             #            25                                                               - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 27 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                - Ala Pro Val Ser Val Gly Gly Gly Thr Val Le - #u Ala Lys Met Tyr Pro         #                15                                                           - Arg Gly Asn His Trp Ala Val Gly His Leu Me - #t                             #            25                                                               - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - Glu Gln Arg Leu Gly Asn Gln Trp Ala Val Gl - #y His Leu Met                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                - Trp Ala Val Gly His Leu Met                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 40 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - Ala Gln Pro Tyr Pro Gln Gly Asn His Glu Al - #a Gln Pro Tyr Pro Gln         #                15                                                           - Gly Asn His Glu Ala Gln Pro Tyr Pro Gln Gl - #y Asn His Glu Ala Gln         #            30                                                               - Pro Tyr Pro Gln Gly Asn His Glu                                             #        40                                                                   __________________________________________________________________________

We claim:
 1. An isolated peptide comprising an amino acid sequence oftyrosine-proline-glutamine-glycine-asparagine-histidine-(SEQ ID NO: 2).2. An isolated peptide having an amino acid sequence selected from thegroup consisting of:AQPYPQGNHEATSSSF (SEQ ID NO: 4); AQPYPQGNHEATSSS(SEQ ID NO: 5); AQPYPQGNHEA (SEQ ID NO: 6); AQPYPQGNHEAT (SEQ ID NO: 7);AQPYPQGNHEATS (SEQ ID NO: 8); AQPYPQGNHEATSS (SEQ ID NO: 9);AQPYPQGNHEATSY (SEQ ID NO: 10); AQPYPQGNHEAAYG (SEQ ID NO: 11);AQPYPQGNHEAAY (SEQ ID NO: 12); AQPYPQGNHEAA (SEQ ID NO: 13); AQPYPQGNHE(SEQ ID NO: 14); AQPYPQGNHEASYG (SEQ ID NO: 15); AQPYPQGNHEASY (SEQ IDNO: 16); AQPYPQGNHEAS (SEQ ID NO: 17); QPYPQGNHEA (SEQ ID NO: 18);AQPYPQGNH (SEQ ID NO: 19); QPYPQGNHE (SEQ ID NO: 20); PYPQGNHEA (SEQ IDNO: 21); QPYPQGNH (SEQ ID NO: 22); PYPQGNHE (SEQ ID NO: 23); YPQGNHEA(SEQ ID NO: 24); PYPQGNH (SEQ ID NO: 25); and YPQGNHE (SEQ ID NO: 26);YPQGNHEATSSSF (SEQ ID NO: 27); YPQGNHEATSSS (SEQ ID NO: 28); YPQGNHEATSS(SEQ ID NO: 29); YPQGNHEATS (SEQ ID NO: 30); and YPQGNHEAT (SEQ ID NO:31).
 3. A pharmaceutical composition comprising the peptide of claim 2and a pharmaceutical carrier or excipient.
 4. An isolated proteincomprising the peptide AQPYPQGNHEASYG (SEQ ID NO: 15).
 5. An isolatedmitogenic protein that has an estimated molecular weight of about 45kDa, said estimate obtained by electrophoresing the HPLC-purifiedprotein on an SDS-polyacrylamide gel, and that has an amino acidsequence beginning at position 1 of the amino terminal end of theprotein, said sequence comprising SEQ ID NO:
 2. 6. The isolatedmitogenic protein of claim 5, wherein said amino acid sequence comprisesalanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-X-X-tyrosine-glycine (SEQ ID NO: 1).
 7. A pharmaceuticalcomposition comprising the protein of claim 5 and a pharmaceuticalcarrier or excipient.
 8. The isolated mitogenic protein of claim 6,further defined as released by BSC-1 cells in culture by wounding.
 9. Apharmaceutical composition comprising the protein of claim 8 and apharmaceutical carrier or excipient.
 10. An isolated mitogenic proteinthat has an estimated molecular weight of about 22 kDa, said estimateobtained by electrophoresing the HPLC-purified protein on anSDS-polyacrylamide gel, and that has an amino acid sequence beginning atposition 1 of the amino terminal end of the protein, said sequencecomprising SEQ ID NO:
 2. 11. The isolated mitogenic protein of claim 10,wherein said amino acid sequence comprisesalanine-glutamine-proline-tyrosine-proline-glutamine-glycine-asparagine-histidine-glutamicacid-alanine-threonine-serine-serine-serine-phenylalanine (SEQ ID NO:4).
 12. The isolated mitogenic protein of claim 10, further defined asreleased by BSC-1 cells in culture by wounding.